Methods of producing doxorubicin

ABSTRACT

The present invention provides novel methods for producing doxorubicin using daunomycin as a substrate. One method employs a genetically engineered host microorganism which is transformed with a vector, preferably a plasmid, which contains the doxA gene. Preferably, the doxA gene, also referred to herein as &#34;doxA&#34;, is cloned into a plasmid which is then introduced into the host microorganism, preferably a bacterial host, more preferably Streptomyces, to provide a transformed host microorganism. The doxA gene, when present on a plasmid, confers on the transformed host the ability to convert daunomycin and 13-dihydrodaunomycin, to doxorubicin. The doxA gene encodes a P450-like enzyme which catalyzes the hydroxylation of daunomycin and 13-dihydrodaunomycin at C-14 to form doxorubicin; such enzyme is designated &#34;daunomycin C-14 hydroxylase&#34;. Thus, the expression of doxA in the transformed host using a plasmid which contains doxA enables the transformed host to convert daunomycin to doxorubicin. The doxorubicin is then extracted from host microorganism cultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of the following commonly assigned,copending U.S. Pat. application Ser. No. 08/653,650, filed May 14, 1996.

BACKGROUND OF THE INVENTION

Daunomycin and doxorubicin are clinically important chemotherapeuticagents. Daunomycin is used primarily to treat adult myelogenousleukemia. Doxorubicin is widely used to treat a variety of neoplasias,making it the more valuable of the two anticancer drugs. The world widemarket for doxorubicin is estimated to exceed $156 million. As of 1984,the wholesale price for doxorubicin was estimated to be $1,370,000 perkilogram.

While daunomycin is synthesized by several species of Streptomyces,doxorubicin is biologically synthesized by only one strain, a mutantstrain of Streptomyces peucetius, called S. peucetius subsp. caesiuswhich is available from the American Type Culture Collection underAccession number 27952.

The alternative in vitro laboratory synthesis of doxorubicin isdifficult. The in vitro synthesis of doxorubicin is a process involvingmultiple steps and resulting in a poor yield, with a lack ofstereospecificity in several of the synthetic steps, producing formswhich are difficult to separate.

Chemical synthetic procedures are known for converting daunomycin todoxorubicin; however they require the use of halogens in the syntheticprocess.

It would be desirable to have an efficient, cost-effective method forproducing doxorubicin that does not require the use of halogens in thesynthetic process.

SUMMARY OF THE INVENTION

The present invention provides novel methods for producing doxorubicinusing daunomycin as a substrate. One method employs a geneticallyengineered host microorganism which is transformed with a vector,preferably a plasmid, which contains the doxA gene. Preferably, the doxAgene, also referred to herein as "doxA", is cloned into a plasmid whichis then introduced into the host microorganism, preferably a bacterialhost, more preferably Streptomyces, to provide a transformed hostmicroorganism. The doxA gene, when present on a plasmid, confers on thetransformed host the ability to convert daunomycin and13-dihydrodaunomycin, to doxorubicin. The doxA gene encodes a cytochromeP450-type enzyme which catalyzes the hydroxylation of daunomycin and13-dihydrodaunomycin at C-14 to form doxorubicin; such enzyme isdesignated "daunomycin C-14 hydroxylase". Thus, the expression of doxAin the transformed host using a plasmid which contains doxA enables thetransformed host to convert daunomycin to doxorubicin. The doxorubicinis then extracted from host microorganism cultures.

Another method for producing doxorubicin involves incubating thedaunomycin C-14 hydroxylase with daunomycin, then extracting thedoxorubicin from the solution.

Another method involves adding daunomycin to cultures of Streptomycessp. strain C5 and extracting doxorubicin from the culture fluid and thehost cells.

The invention also relates to daunomycin C-14 hydroxylase, novelplasmids, novel polylinkers and novel transformed host microorganismsemployed in such method for producing doxorubicin. The invention alsorelates to methods for producing anthracyclines, such as13-deoxycarminomycin and 13-deoxydaunomycin, 13-dihydrocarminomycin and13-dihydrodaunomycin, carmninomycin and daunomycin.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a restriction map of the Streptomyces sp. strain C5 daunomycinbiosynthesis gene cluster which shows the position of doxA within thecluster. Abbreviations for restriction endonuclease sites are asfollows: "B" represents BamHI; "Bg" represents BglII; "C"representsClaI; "E" represents EcoRI; "K" represents KpnI; "P" represents PstI;"S" represents SstI; and "X" represents XhoI;

FIG. 2 is a detailed restriction map of part of the daunomycinbiosynthesis gene cluster from Streptomyces sp. strain C5. Abbreviationsfor restriction endonuclease sites are as follows: "B" represents BamHI;"Bg" represents BglII; "E" represents EcoRI; "K" represents KpnI; "P"represents PstI; "S" represents SstI; "Sp" represents SphI;

FIG. 3A and 3B is a nucleotide sequence of the 3196 base pair KpnI- SstIDNA fragment from Streptomyces sp. strain C5 containing the doxA gene.The deduced amino acid sequence of the daunomycin C-14 hydroxylase isgiven below the nucleotide sequence. Potential ribosome binding sites,designated "rbs" are identified, as are significant restrictionendonuclease sites. The sequences and deduced products of the 3' end oforfl, all of orfA, and the 5' end of dauI are also shown;

FIG. 4 shows the plasmid maps of plasmid pANT849 and the plasmids pANT42and pANT842 which were used to construct pANT849;

FIG. 5 shows the plasmid maps of pANT195 and plasmids pANT849, pANT186,pANT185, pANT235 and pUC19 all of which were used to construct pANT195;

FIG. 6 shows the sequence of snpR, doxA, and the intervening sequenceswithin plasmid pANT195;

FIG. 7 shows plasmid maps of pANT192 and pANT193;

FIG. 8 shows plasmid maps of pANT194 and pANT196;

FIG. 9 shows the N-terminal, modified region of the doxA fusion protein;

FIG. 10 shows plasmid maps of pANT198 and pANT199; and

FIG. 11 shows sequence of the doxA gene and upstream melC1 promoterregeion in pANT196; and

FIG. 12 shows a plasmid map of pANT144.

DETAILED DESCRIPTION OF INVENTION

A novel method for producing doxorubicin from daunomycin has beendeveloped which employs genetically engineered host microorganisms thatcontain and express the gene doxA. Preferably, doxA is cloned into aplasmid which is then inserted into a host microorganism, preferably abacterial host, more preferably Streptomyces, most preferablyStreptomyces lividans to provide a transformed host. The doxA geneencodes daunomycin C-14 hydroxylase which catalyzes the hydroxylation ofdaunomycin and 13-dihydrodaunomycin at C-14 to form doxorubicin. Thus,expression of doxA in the transformed host using a plasmid whichcontains the doxA gene enables the transformed host to convertdaunomycin to doxorubicin.

Daunomycin is also known as daunorubicin; doxorubicin is also known as14-hydroxydaunomycin and adriamycin. The structure of daunomycin isshown below: ##STR1##

The structure of doxorubicin is shown below: ##STR2## Cloning andAnalysis of the DoxA Gene

Streptomyces sp. strain C5 synthesizes several compounds infermentations, that is such compounds are produced from common metabolicintermediates and without the addition of precursor anthracyclinemolecules to the culture media. Streptomyces sp. strain C5 produces thefollowing anthracyclines: e-rhodomycinone; daunomycin;13-dihydrodaunomycin; baumycin A1; and baumycin A2. Nevertheless, agene, the doxA gene, was discovered in the genome of Streptomyces sp.strain C5 which, when expressed, converts daunomycin, particularlyexogenous daunomycin, to doxorubicin. Preferably, the conversion ofdaunomycin to doxorubicin is accomplished by cloning the doxA gene alongwith a promoter into a plasmid which is then introduced into a hostmicroorganism.

It has also been discovered that Streptomyces sp. strain C5 can convertsmall amounts, less than 10%, daunomycin to doxorubicin in the absenceof plasmid containing the doxA gene.

Preferably, the doxA gene is cloned from Streptomyces, preferablyStreptomyces sp. strain C5. Alternatively, the doxA gene is synthesizedusing conventional oligonucleotide synthesis techniques and equipment.

The doxA gene is located in the daunomycin biosynthesis gene clusterbetween the daunomycin polyketide biosynthesis genes and dauI, aputative transcriptional activator as shown in FIG. 1. The location ofdoxA within the Streptomyces sp. strain C5 daunomycin biosynthesis genecluster is shown in FIG. 2.

The approximately 8 kbp region between daul, a gene encoding anactivator regulatory protein for daunomycin biosynthesis, and thedaunomycin polyketide synthase biosynthesis genes was sequenced in itsentirety.

Plasmids containing inserts to be sequenced were isolated fromrecombinant E. coli JM83, available from Dr. Mary Berlyn, E. coliGenetic Stock Center, Yale University, P.O. Box 6666, New Haven, Conn.06511-7444 by the methods disclosed in Carter, M. J., and I. D. Milton,(1993), "An Inexpensive and Simple Method for DNA Purification on SilicaParticles," Nucleic Acid Res. Volume 21, p. 1044. The doxA DNA wassequenced in both directions, that is, both strands were sequenced usingSequenase enzyme, Version 2.0 from the United States Biochemical Corp.,Cleveland, Ohio, according to the manufacturer's instructions, and asdescribed in Ye, et. al., 1994, "Isolation and Sequence Analysis ofPolyketide Synthase Genes from the Daunomycin-producing Streptomyces sp.strain C5 " J Bacteriol. 176:6270-6280. Doubled-stranded DNA templateswere employed. The terminated chains were labeled with 3000 Ci/mmol (a-³² P)dCTP from Dupont -New England Nuclear, Boston, Mass. The terminatedlabeled chains were separated on a 6% weight-to-volume polyacrylamidegel containing 10% (volume-to-volume) formamide and visualized byautoradiography. Sequencing reactions were carried out using7-deaza-dGTP nucleotide mixes to reduce compressions. Forward (-40) andreverse universal pUC/m13 17-mer oligonucleotide primers from U.S.Biochemical Corp. were used to obtain the initial sequences in theinserts. Specific primers, 15-mer oligonucleotides, were generated basedon sequencing results for extension of the sequences within the inserts.

DNA sequence data were analyzed using Clone Manager from Stateline, Pa.,and the Sequence Analysis Software Package of the Genetics ComputerGroup from Madison, Wis.

The nucleotide sequence between dauI and the ketoreductase justdownstream of dauA-orfG is shown in FIG. 3. Two complete open readingframes, orfA and doxA, were found within this sequence; OrfA encodes aprotein of M_(r) 28,808, and 275 amino acid residues, and doxA encodes aprotein of M_(r) 46,096 and 422 amino acid residues.

Plasmids

The doxA gene is inserted into a vector, preferably a plasmid.Optionally, the plasmid contains genes from the daunomycin synthesiscluster in addition to doxA. However, preferred plasmids lack dauA(g)and more preferred plasmids lack dauA(g), orfl and orfA.

The preferred plasmids contain not only the translated portion of doxAbut a promoter. Suitable promoters include, Streptomyces promoters forexample, melC1-P, ermE-P, wild type, and snpA-P. The snpA-P promoter isthe most preferred. Preferably, the promoter is a protein activatedpromoter, and most preferably, an SnpR-activated promoter. Lesspreferred plasmids, such as pANT196, contain a melC1 promoter frompIJ702 for expression of doxA. Also less preferred are plasmids whichlack a known promoter, such as pANT194.

The most preferred plasmid which contains doxA is designated "pANT195"which is shown in FIG. 5. Host microorganisms, when transformed withplasmid pANT195, convert 100% daunomycin to doxorubicin. Other plasmidswhich contain doxA are suitable, including, for example pANT192,pANT193, pANT194 and pANT196. Host microorganisms, when transformed withplasmid pANT192, typically convert about 25% daunomycin to doxorubicinat a concentration of 2 mg/ml. Host microorganisms when transformed withpANT193 convert about 80% daunomycin to doxorubicin and about 20%daunomycin to 13-dihydrodaunomycin, at a daunomycin concentration of 2mg/ml.

Construction of the Plasmids

Digestion of and ligation of DNA was performed using conventionaltechniques described by Maniatis et al. (1982) in "Molecular Cloning: ALaboratory Manual," Cold Spring Harbor Laboratory, New York.

Construction of pANT195

Plasmid pANT195, shown in FIG. 5, has about 7.04 kbp of DNA. PlasmidpANT195 was constructed by inserting the 1.72 kbp SphI-SacI fragmentinsert containing intact doxA from plasmid pANT186 into pANT849.

First, plasmid pANT186 was constructed by constructing pANT235. PlasmidpANT235 is described in Ye et. al. 1994 "Isolation and Sequence Analysisof Polyketide Synthase Genes from the Daunomycin-Producing Streptomycessp. Strain C5 " J Bacteriol. Vol. 176, pp. 6270-6280. Plasmid pANT235 isa 9.2 kbp plasmid which contains a 6.48 kbp BamHI-BglII DNA fragmentfrom the Streptomyces sp. strain C5 daunomycin biosynthesis genecluster. The doxA gene lies within the insert of pANT235 which isderived from the daunomycin biosynthesis gene cluster. The BamHI-BglIIDNA fragment had been cloned into the BamHI site of pUC19 to generatepANT235. Plasmid pUC19 is available from Gibco BRL, Gaithersburg Md.

Next, pANT235 was digested with SalI and SstI and the digestion productswere purified on an agarose gel. The 1.67 kbp SalI-SstI fragmentcontaining the 3₋₋ end of the doxA gene and the 5₋₋ end of daul wasextracted from the agarose gel and ligated into pUC19 with T4 DNAligase, from Gibco BRL, Gaithersburg Md., to generate pANT185, as shownin FIG. 5.

Next, pANT235 was used as the template for the polymerase chain reactionamplification of the 5₋₋ -end of the doxA gene containing an upstreamribosome binding site and SphI restriction site for the 5' end and BspEIrestriction site for the 3' end.

The forward primer used in the polymerase chain reaction amplificationof the doxA gene had the following nucleotide sequence: 5₋₋-GACATGCATGCGGAGGGGTGCCTC-3₋₋ SEQ ID NO:1 The forward primer which isused for the 5₋₋ -end, contains an SphI site with five extra nucleotideson the end and the extra ribosome binding site "GGAGG". The reverseprimer had the following nucleotide sequence: 5₋₋-GACGCAGCTCCGGAACGGGG-3₋₋ SEQ ID NO:2 The reverse primer which is usedfor the 3₋₋ -end, has a BspEI site plus eight extra nucleotides.

The polymerase chain reaction amplification was carried out for 25cycles using Deep Vent Polymerase from New England Biolabs, Beverly,Mass. The solution for PCR included: 2.0 ml dimethylsulfoxide; 14.5 mldouble distilled water; 1.25 mM dNTPs, 16.0 ml of a total stockcontaining DATP, dCTP, dTTP, dGTP; 5.0 ml 10×Deep Vent Buffer, from NewEngland Biolabs; 5 ml forward primer; 5 ml reverse primer; 0.5 ml DeepVent polymerase; 2.0 ml DNA template, 14.5 ml distilled water, andboiled for 10 minutes. PCR was carried out by incubating the reactionmixture at 94° C. in the absence of Deep Vent Polymerase for 5 minutes,following by 25 cycles of the following regimen: 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. for 30 seconds. After 25 cycles werecompleted, the mixture was incubated at 72° C. for 7 minutes and thenheld at 4° C. until further use.

The products of PCR were separated on a 0.8% agarose gel, and a 298 basepair DNA fragment was eluted from the gel. The 298 base pair DNAfragment was then digested with SphI and BspEI to generate a 285 basepair fragment with "sticky" ends. pANT185 was digested with SphI andBspEI and the 285 base pair fragment ligated into pANT185 to generatepANT186 which was introduced into dam/dcm-minus E. coli strain ET12567.MacNeil, et. al. (1992) "Analysis of Streptomyces avermitilis GenesRequired for Avermectin Biosynthesis Utilizing a Novel IntegrationVector", Gene, vol. 111, pages 61-68. Plasmid pANT186 contains thecomplete doxA gene, upstream of which lay the newly constructed ribosomebinding site having the nucleotide sequence GGAGG. The nucleotidesequence of the PCR-generated 5₋₋ end of the gene was confirmed bydideoxy sequencing.

Plasmid pANT849, shown in FIG. 4, was constructed by first constructingpANT842. Plasmid pANT42, described in Lampel, et. al., 1992 "Cloning andSequencing of a Gene Encoding a Novel Extracellular Neutral Proteinasefrom Streptomyces sp. Strain C5 and Expression of the Gene inStreptomyces lividans 1326 "J. Bacteriology 174:2797-2808, was digestedwith KpnI and religated, removing a 1.95 kbp KpnI fragment to yieldpANT842.

A novel polylinker sequence, having 48 nucleotides, was constructedaccording to conventional techniques using synthesized DNAoligonucleotides by Integrated DNA Technologies, Inc., Coralville, Iowa.The polylinker sequence has the following nucleotide sequence:

    SEQ ID NO:3    SphI BglIISacI DraI HpaI    GCATGCGAATTCAGATCTAGAGCTCAAGCTTTAAACTAGTTAACGCGT      EcoRI XbaI HindIII SpeI MluI

Plasmid pANT842 was digested with SphI-MluI to remove a 1.42 kbpSphI-MluI fragment. The polylinker sequence was ligated intoSphI-MluI-digested pANT842 to provide plasmid pANT849. Plasmid pANT849,shown in FIG. 4, has 5.34 kbp of DNA and lacks the snpA gene and most ofmelC2. Plasmid pANT849 does have the SnpR-activated snpA-promoter, whichis located immediately upstream of the polylinker sequence as shown inFIG. 4. pANT849 is a high copy number plasmid and contains thethiostrepton resistance gene as the selectable marker.

Next, to construct pANT195, a clone of pANT186 which contains themodified doxA gene, was digested with SphI and SstI, and pANT849 wasdigested with SphI and SstI. The fragment from pANT186 containing thedoxA gene was ligated into the polylinker sequence of pANT849 to makepANT195 as shown in FIG. 5.

The sequence of the region of pANT195 containing the snpR activatorgene, the SnpR-activated snpA promoter, and the 5₋₋ -end-modified doxAgene is shown in FIG. 6, and SEQ ID NO:6.

Construction of pANT192

Plasmid pANT192 shown in FIG. 7 is an 11.84 kbp plasmid which containsDNA encoding the acyl carrier protein and its putative promoter, aketoreductase (orf1), orf2, a partial orf3, orfA, doxA, dauI, and mostof dauJ. Plasmid pANT192 was constructed by removing the 6.52 kbpHindIII-EcoRI fragment from pANT235 which includes the entireBglII-BamHI fragment, by digesting pANT235 with HindIII and EcoRI. NextpANT849 was digested with HindIII and EcoRI and the 6.52 kbpHindIII-EcoRI fragment from pANT235 was ligated into pANT849.

Construction of pANT193

Plasmid pANT193, shown in FIG. 7, has 10.28 kbp of DNA and contains partof orf1, all of orfA, and doxA driven by the snpR-activatedsnpA-promoter. Plasmid pANT193 was constructed by digesting plasmidpANT235 with KpnI, and the 1582 base pair KpnI fragment removed. Theplasmid was re-ligated to itself to form pANT235-k. Plasmid pANT235-kwas digested with EcoRI and HindIII to remove the 4.95 kbp EcoRI-HindIIIfragment. pANT849 was digested with EcoRI and HindIII and the 4.95 kbpEcoRI-HindIII fragment from pANT235-k was ligated into the digestedpANT849.

Construction of pANT194

Plasmid pANT194, shown in FIG. 8, has 8.97 kbp of DNA and contains thepart of orf1, all of orfA, daul, dauJ and doxA but lacks any knownpromoter to drive the expression of doxA. Plasmid pANT194 wasconstructed by digesting pANT192 with KpnI to remove a 2.87 kbp KpnIfragment and then religating the plasmid to itself.

Construction of pANT196

Plasmid pANT196, shown in FIG. 8, has 7398 bp of DNA and possesses apromoter melC1 which drives the expression of the doxA gene. pANT186 wasdigested with SphI and SstI and a 1712 nucleotide SphI-SstI fragmentfrom pANT186 containing doxA was isolated and ligated into SphI-SstIdigested pIJ702. Plasmid pIJ702 is a 5.686 kbp plasmid which isdescribed in Katz, E., et. al. (1983) "Cloning and Expression of theTyrosinase Gene from Streptomyces antibioticus in Streptomyces lividans"J. Gen. Microbiol. volume 129, pages 2703-2714.

Construction of pANT198

Plasmid pANT186 was digested with SphI and then incubated with T4 DNApolymerase from Gibco BRL, according to the manufacturer's instructionsto yield a blunt end. Plasmid pZero from Invitrogen, San Diego, Calif.,was digested with EcoRI and then filled in 5' to 3' using Klenowfragment of DNA polymerase according to the manufacturer's instructionsto provide a blunt end. Both fragments were purified according to themethods described in Carter, M. J. and I. D. Milton (1993), Nucleic AcidRes. volume 21, pages 1044. The fragments were precipitated in ethanolfor one hour at -70° C. and then digested with SstI overnight. Theplasmid and insert, each of which contains a single blunt end and anSstI end, were purified from an agarose gel and then ligated overnightwith T4 DNA ligase at room temperature, to provide pANT198.

Construction of pANT199

Plasmid pANT198 was digested with EcoRI-HindIII, the EcoRI-HindIIIfragment removed and ligated into pTrcHisC from Invitrogen to constructpANT199. In pANT199, the doxA gene is translationally fused with aleader sequence encoding six histidine residues, shown in SEQ ID NO:7 sothat the fusion protein can be affinity purified on a nickel-agarosegel.

pANT849

Plasmid pANT849 in addition to being useful to construct pANT195 is alsouseful expression vector for other genes. To construct other suchplasmids pANT849 is digested with at least one restriction endonucleasecorresponding to the restriction sites in the polylinker, such as, forexample, SphI, BglII, SacI, DraI, HpaI, EcoRI, XbaI, HindIII, SpeI, orMluI. The desired gene sequence to be inserted into the plasmid isprovided with sticky ends corresponding to the sticky ends of the cutpANT849. The desired gene is then ligated into the plasmid to provide anew plasmid derived from pANT849.

Host Microorganisms

Suitable host microorganisms for the doxA plasmid possess electrondonating, cytochrome P450 accessory proteins; suitable accessoryproteins include for example, NADPH:ferredoxin oxidoreductase andferredoxin. The preferred host microorganisms are bacteria, morepreferably E. coli or Streptomyces spp., most preferably Streptomyceslividans TK24 and Streptomyces coelicolor CH999. Streptomyces coelicolorCH999 is a mutant of Streptomyces coelicolor A3(2). Streptomyceslividans TK4 is available from Professor David A. Hopwood, Head,Department of Genetics, John Innes Centre, Norwich Research Park,Colney, Norwich NR4 7UH, United Kingdom. Streptomyces coelicolor CH999is available from C. Khosla, Stanford University, Palo Alto, Calif. andProfessor David A. Hopwood. S. peucetius is available from the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., U.S.A.under the accession number 29050. Streptomyces sp. strain C5 wasobtained from the Frederick Cancer Research Center, Frederick Md.Streptomyces sp. strain C5 is also available from the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., U.S.A. underthe Accession number ATCC

Transformation of Host Microorganism

The plasmids are introduced into the host microorganism usingconventional techniques. For example, Streptomyces spp. are transformedusing electroporation as described in Pigac and Schrempf (1995) "ASimple and Rapid Method of Transformation of Streptomyces rimosus R6 andOther Streptomycetes by Electroporation", Appl. Environ. Microbiol. vol.61, pages 352-356, or by protoplast transformation. Streptomyces arepreferably transformed using protoplast transformation as described inHopwood, et. al. (1985), "Genetic Manipulation of Streptomyces: ALaboratory Manual", The John Innes Foundation, Norwich, UK. E. colistrains are transformed using conventional transformation procedures asdescribed in Maniatis, et al. (1982) "Molecular Cloning: A LaboratoryManual" Cold Spring Harbor Laboratory, New York.

Plasmid pANT195 was introduced into Streptomyces lividans TK24 byprotoplast transformation according to the procedures described inHopwood et. al (1985), "Genetic Manipulation of Streptomyces: ALaboratory Manual", The John Innes Foundation, Norwich, UK. 500 ml ofStreptomyces lividans TK24 protoplasts, were transformed with 10 ml ofplasmid DNA, about 0.5 mg total, in 500 ml of T buffer for two minutes.The reaction was stopped with 500 ml of P buffer and the protoplastswere pelleted twice in a microcentrifuge for 7 seconds each spin. Thepellets were then resuspended in 100 ml of P buffer and plated onto R2YEmedium using a soft R2YE agar overlay with 50 mg/ml of thiostreptonadded 24 hours later. The transformed microorganisms were tested fortheir ability to carry out daunomycin C-14 oxidation.

S. peucetius ATCC 29050, Streptomyces coelicolor CH999 and Streptomycessp. strain C5 were transformed with plasmids pANT195 and pANT849 byprotoplast transformation.

Daunomycin C-14 Hydroxylase

The daunomycin C-14 hydroxylase encoded by doxA is a cytochromeP450-type enzyme having a deduced Mr of 46,096. Daunomycin C-14hydroxylase is a monooxygenase which inserts a single oxygen at carbon14 on daunomycin. The daunomycin C-14 hydroxylase also appears tocatalyze the two step oxidation at C-13 from methylene to hydroxyl to aketo functional group. Daunomycin C-14 hydroxylase also oxidizes13-dihydrocarminomycin to carminomycin and 13-dihydrodaunomycin todoxorubicin.

The deduced amino acid sequence of daunomycin C-14 hydroxylase which isencoded by doxA of strain C5 is shown in FIG. 3 and SEQ ID NO:5.

Preparation of Daunomycin C-14 Hydroxylase

EXAMPLE A

The daunomycin C-14 hydroxylase was isolated and partially purified andsubjected to spectrophotometric analysis. First, S. lividans TK24strains containing plasmid pANT195 were grown in YEME medium containing10 mg/ml thiostrepton for 48 hours at 30° C., harvested and washed bycentrifugation and then broken in 100 mM, pH 7.5 sodium phosphate bufferusing a French pressure cell at 15,000 lb/in². The cell debris andunbroken mycelia were pelleted by centrifugation at 10,000'g for 30minutes at 4° C., after which the supernatant was analyzed by visiblespectrometry. The cytochromes within the supernatant derived from thecultures were reduced by a few grains of sodium dithionite. Thesupernatant samples were bubbled with carbon monoxide for 1 minute priorto analysis. Spectra were obtained using a Beckman DU-64 single beamspectrophotometer and reduced-plus-CO minus reduced difference spectrawere obtained by electronic subtraction.

Reduced-plus-CO minus reduced difference spectra of samples derived fromcultures of S. lividans TK24 containing plasmid pANT195 revealed a peakat 450 nm, characteristic of cytochrome P450 enzymes. Such peak was notobserved in samples derived from the control culture, which lack thedoxA gene.

Proteins derived from both S. lividans TK24 containing plasmid pANT195and the control culture S. lividans TK24 containing control plasmidpANT849, were visualized by sodium dodecylsulphate polyacrylamide gelelectrophoresis. Samples derived from cultures containing plasmidpANT195 revealed a band with M_(r) of about 42,000, close to thepredicted size of daunomycin C-14 hydroxylase. This band was not presentin samples derived from the control cultures.

EXAMPLE B

A 50 ml culture of S. lividans TK24 (pANT195) was grown for 48 hours inYEME medium plus 10 mg/ml thiostrepton as in Example 1. This culture wassplit into 2×25ml aliquots, each of which was used to inoculate a 1000ml flask containing 225 ml of YEME medium plus 10 mg/ml thiostrepton,giving 2 fresh 250 ml cultures, which were grown as described in Example1 for 48 hours. A 14-liter stirred tank fermentor containing 9.5 litersof YEME medium with 10 mg/ml of thiostrepton was inoculated with both250 ml cultures, a total inoculum size of 500 ml, and the 10 L culturewas incubated for 6 days under the following conditions: temperature,28° C.; air flow, 1 volume air/volume culture/minute; agitation, 250rpms. The culture was harvested by continuous centrifugation using aHeraeus 300 MD System, from Heraeus Sepatech, South Plainfield, N.J., at15,000 rpm and a flow rate of 100 ml/min. The resultant pellet wasfrozen at -70° C. until further use. A small portion of the frozenpellet of S. lividans TK24 (pANT195) was thawed on ice in ice-cold 0.1 Msodium phosphate (Na₂ HPO₄ :NaH₂ PO₄) buffer having a pH 7.5. The thawedsuspension was passed twice through a 4° C. French Pressure cell at15,000 pounds per square inch to break the cells. The broken cellsuspension was centrifuged at 10,000×g for 30 minutes at 4° C. and thesupernatant from this centrifugation step was kept on ice to provide anisolated partially purified daunomycin C-14 hydroxylase.

P450 Determination

A 100 ml aliquot of the daunomycin C-14 hydroxylase prepared accordingto Example B, was added to 900 ml of 0.1M sodium phosphate buffer havinga pH of 7.5, in a cuvette and approximately 1 mg of sodium dithionitewas added to reduce the sample. This sample was used as the backgroundfor a spectrophotometric scan from 400 nm to 600 nm. Carbon monoxide wasbubbled through this sample for one minute and the sample was scannedagain from 400-600 nm. Electronic subtraction of the reduced plus carbonmonoxide minus reduced sample revealed a sharp peak at 450 nm,indicative of the active cytochrome P450 enzyme. This assay was usedbefore, during and after Examples 19 to 26 to ensure that the daunomycinC-14 hydroxylase was active and stable. In all cases, the cytochromeP450 activity appeared to be 100% of the original.

Method C

The Fusion Protein

Plasmid pANT199 was introduced by transformation into E. coli strainTOP10 from Invitrogen. Transformants were selected using ampicillin andgrown in 3.0 ml cultures of SOB medium overnight at 37° C. The recipefor the SOB medium was provided by Invitrogen. Fifty mL of this culturewas used to inoculate 3.0 ml of fresh SOB medium. The new culture wasgrown at 37° C. for 2 hours to an optical density of 0.6 and theninduced with IPTG at 1.0 mM final concentration for 5 hours. The culturewas then harvested by centrifugation in a microcentrifuge and the pelletwas frozen overnight at -20° C. The next day the pellet was boiled inSDS-PAGE sample buffer described in Laemmli, U.K. (1970) "Cleavage ofStructural Proteins during the assembly of the head of Bacteriophage T4"Nature volume 227, pages 680-685, and run on a 10% (w/v) SDS-PAGE gel. Aprotein with M_(r) of about 52,000 was observed that wasinsert-specific, the approximate size expected for the fusion proteinbased on amino acid sequence.

The fusion protein is then bound to a nickel-agarose column fromInvitrogen, Inc., San Diego, Calif., and washed with 50 mM sodiumphosphate buffer at pH 8.0 containing also 300 mM NaCl and 20 mMimidazole. The protein is then eluted using the same buffer butcontaining with 250 mM imidazole buffer at pH of 8, to provide a purefusion protein with a modified N-terminus as shown in FIG. 9 and SEQ IDNO:8 . The leader sequence is then cleaved from the fusion protein usingenterokinase available from Biozyme Lab. Int'l Ltd. San Diego Calif.,according to the manufacturer's directions, to provide pureN-terminal-modified daunomycin C14 hydroxylase as shown in SEQ ID NO:9.

Methods of Converting Daunomycin to Doxorubicin

A host microorganism transformed with a plasmid containing the doxA geneis grown preferably in liquid culture, and daunomycin is added to theculture broth. Preferably, the daunomycin is added at a concentration offrom about 2 mg/L to 22.2 mg/L, more preferably about 2 to 10 mg/L.Preferably, the daunomycin concentration is below about 10 mg/L. Wherethe concentration is above about 10 mg/L, the daunomycin tends to killthe host microorganisms although doxorubicin is still produced. Theculture of transformed host microorganism is then incubated with thedaunomycin; the longer the incubation the greater the amount ofdaunomycin is converted to doxorubicin. Preferably, the culture isincubated at least 6 hours, more preferably, at least 24 hours with thedaunomycin.

A 48 hour culture has sufficient biomass to convert 2 mg/L daunomycin todoxorubicin within 24 hours.

Next, the doxorubicin is extracted preferably from both the transformedmicroorganisms and the culture fluid, using conventional techniques. Asuitable technique involves extracting the transformed microorganismsand the culture fluid, preadjusted to a pH of about 8.5, with a mixtureof chloroform and methanol and separating and drying the organic phaseto provide a culture extract. The culture extract is resuspended inmethanol and the components of the culture extract are separated,preferably by chromatography, to provide doxorubicin.

Media Composition

GPS production medium contains: glucose, 22.5 g/L; Proflo from Traders,Memphis, Tenn., 10 g/L; NaCl, 3 g/L; CaCO₃, 3 g/L, and 10 ml/L tracesalts according to Dekleva, M. L. et. al. (1985), "Nutrient Effects onAnthracycline Production by Streptomyces peucetius in a Defined Medium",Canad. J. Microbiol. vol. 31, pages 287-294.

APM seed medium contains the following: yeast extract, 8 g/L; maltextract, 20 g/L; NaCl, 2 g/L 3-(N-morpholino)propanesulfonic acidbuffer, 15 g/L; antifoam B from Sigma Chemical Co., St. Louis, Mo., 4ml/L; 10% weight to volume MgSO₄, 1 ml/L; 1% weight to volume FeSO₄, 1ml/L; 10% weight to volume ZnSO₄, 0.1 ml/L; 50% weight to volumeglucose, 120 ml/L, added after autoclaving; tap water to 1.0 L asdescribed in Guilfoile and Hutchinson, (1991), "A Bacterial Analog ofthe mdr Gene of Mammalian Tumor Cells is present in Streptomycespeucetius, the Producer of Daunorubicin and Doxorubicin", Proc. Nat'l.Acad. Sci. USA volume 88, pages 8553-8557.

The YEME medium contained 3 g/L yeast extract available from U.S.Biohemical Corp. Cleveland, Ohio; 5 g/L bacto-peptone from DifcoDetroit, Mich.; 3 g/L Difco malt extract; 10 g/L glucose; 200 g/Lsucrose; and 2 ml/L of an autoclave-sterilized solution of 2.5 M MgCl₂×6H₂ O. The pH was adjusted to 7.2 and the solution was autoclaved at121° C. for 20 minutes at 15 psi, to provide the YEME medium.

The nitrate-defined-plus-yeast extract medium, also referred to hereinas "NDYE medium", contains the following: yeast extract, 5.0 g/L;N-(2-hydroxyethyl)piperazine-N₋₋ -(2-ethanesulfonic acid) buffer 4.8g/L; 0.06 g/L anhydrous MgSO₄ ; 0.24 g/L K₂ HPO₄ ×3H₂ O; 4.28 g/LNaNO_(3;) 1.0 ml/L 20×trace elements; 45% (w/v) glucose solution, 50ml/L; pH 7.3. The 20×trace elements solution contains the followingelements in double distilled water: ZnCl₂, 800 mg/L; FeCl₃ ×6H₂ O, 4000mg/L; CuCl₂ ×2H₂ O, 40 mg/L; MnCl₂ ×4H₂ O, 40 mg/L; Na₂ B₄ O₃ ×10H₂ O,40 mg/L; (NH₄)₆ Mo₇ O₂₄ ×4H₂ O, 200 mg/L; NiCl, 100 mg/L as described byDekleva et al. (1985) Can. J. Microbiol volume 31, pages 287-294. A fewdrops of Mazu DF60-P antifoam, obtained from Mazer Chemical Co., Gurney,Ill., were added to control foaming in cultures containing NDYE medium.Other antifoam agents, such as Sigma Antifoam B from Sigma ChemicalCompany, are also suitable.

EXAMPLES

The following examples are illustrative and not intended to be limiting.

Methods of Producing Doxorubicin Employing Host Microorganisms

Example 1

Cultures of S. lividans TK24 containing plasmid pANT195 and controlcultures of S. lividans TK24 containing plasmid pANT849 were grown onR2YE agar medium containing 10 mg/ml of thiostrepton in standard 100mm×15 mm plastic petri dishes for 5 days at 30° C., at which time theentire cultures had sporulated. The spores from one entire petri platewere used to inoculate 50 ml of modified YEME medium in 250 mlerlenmeyer flasks. Then 10 mg/ml thiostrepton in DMSO were added to themedium. The thiostrepton was added as selective pressure to maintain theplasmids. The cultures were grown for 48 hours at 30° C. with rotaryshaking at 250 rpm, one inch throw on the shaker. After 48 hours, a 1.5ml sample was removed for plasmid analysis to ensure the presence andsize of the insert DNA containing the doxA gene using a "mini-prep"procedure according to Carter, M. J., and I. D. Milton, (1993), "AnInexpensive and Simple Method for DNA Purification on Silica Particles,"Nucleic Acid Res., Vol. 21, page 1044. Restriction endonucleasedigestion with BspEI and agarose gel electrophoresis generated 1.7 kbp,2.2 kbp, and 3.14 kbp fragments which indicated an intact plasmid andinsert. Then a 10 mL solution containing 100 mg of filter sterilizeddaunomycin-HCl in distilled water was added to the cultures for a finalconcentration of 2.0 mg of daunomycin per ml of culture broth.Incubation was continued for 72 hours.

After a total of 120 hours, that is, 48 hours growth and another 72hours of continued growth in presence of the daunomycin, the pH of theculture was 7.6. The remaining culture broth, a volume of 48.5 ml, wasbrought to pH 8.5 with the dropwise addition of 5 N NaOH. Then the wholeculture broth, including culture fluid and cells, was extracted oncewith a 2×volume of chloroform:methanol at a ratio of 9:1. The organicphase was separated from the aqueous phase by centrifugation at about10,000×g for 10 minutes and then the organic phase was removed by pipet.The organic phase was air dried in a chemical fume hood, thenresuspended in 1 ml of 100% reagent grade methanol and spotted ontoaluminum-backed, 0.25 mm silica gel thin layer chromatography platesfrom Whatman, Clifton, N.J. The components derived from the organicphase culture extract were separated using a solvent system ofchloroform:methanol:acetic acid:water (80:20:16:6). The anthracyclinesin the culture extracts were visualized on the plates by their normalpigmentation and by their fluorescence under ultraviolet irradiation at365 nm. Table 1 shows the results.

The culture extracts of Example 1 and known standards also wereseparated and analyzed by high performance liquid chromatography using aC₁₈ mBondaPak reverse phase column from Waters Corp. Milford, Mass. Thesolutions of standards and culture extracts of Example 1 were filteredthrough 0.2 mm Nylon Acrodisc®13 filters from Gelman Sciences, AnnArbor, Mich. and separated by HPLC using a mobile phase ofmethanol:water (65:35) brought to a pH of 2.5 with 85% phosphoric acidusing a Waters 600E Multisolvent Delivery Pump and Controller and U6K0-2.0 ml manual injector and detected on-line at 254 nm using a Waters486 Tunable Absorbance Detector. The data were analyzed on-line andpost-run using "Baseline 815" software and a 386 SX PC-compatiblecomputer. The products extracted from the cultures were compared tostandards run in parallel and by co-chromatography. These results areshown in Table 2.

                  TABLE 1    ______________________________________    THIN LAYER CHROMATOGRAPHY OF DOXORUBICIN    PRODUCED ACCORDING TO EXAMPLE 1 AS COMPARED TO    KNOWN STANDARDS                                  R.sub.f of                                  Sam-    Sample                        ple    ______________________________________    Daunomycin Standard           0.56    Doxorubicin Standard          0.36    13-Dihydrodaunomycin Standard 0.39    Doxorubicin from Cultures Containing Plasmid doxA (pANT195)                                  0.36    13-Dihydrodaunomycin from Control Culture                                  0.39    ______________________________________     A few grains of each standard was reconstituted in 1 ml of methanol.

As indicated by the results in Table 1, the cultures transformed withplasmid pANT195 containing doxA which were incubated with daunomycin,produced doxorubicin. In contrast, the control cultures produced13-dihydrodaunomycin. Co-chromatography confirmed these results.

                  TABLE 2    ______________________________________    HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF    DOXORUBICIN PRODUCED ACCORDING TO EXAMPLE 1    AS COMPARED TO KNOWN STANDARDS                          Retention time of                          sample.sup.2    Sample                (minutes)    ______________________________________    Daunomycin.sup.1 Standard                          13.3    Doxorubicin.sup.1 Standard                          8.4    13-Dihydrodaunomycin Standard                          10.6    Doxorubicin from doxA    Transformants         4.1; 7.2 (minor)    Control               4.1; 10.6; 21.2    ______________________________________     .sup.1 Standards were reconstituted at 1.0 mg/ml in methanol.     .sup.2 A methanol peak at 2.9 minutes was found in all samples.

The doxA transformants converted greater than 90% of the daunomycin todoxorubicin in 72 hours as evidenced by both TLC and HPLC analyses. Thecontrol cultures, which lack the doxA gene, converted daunomycin to13-dihydrodaunomycin, but not to doxorubicin.

The culture extracts of Example 1 and the standards were hydrolyzed totheir respective aglycones to verify the chemical structures. The acidhydrolysis product of the doxorubicin is adriamycinone, and the acidhydrolysis product of the 13-dihydrodaunomycin is13-dihydrodaunomycinone. The acid hydrolysis product of the doxorubicinproduced by Example 1 was adriamycinone. The acid hydrolysis product ofthe 13-dihydrodaunomycin produced by control cultures of Example 1 was13-dihydrodaunomycinone.

Example 2

The procedure of Example 1 was repeated, with the following exceptions.The cultures were grown at 28° C. rather than 30° C., for 48 hours.After 48 hours of growth, 500 mg of daunomycin-HCl were added to thecultures for a final concentration of 10.0 mg/ml of daunomycin followedby further incubation for 36 hours, instead of 72 hours.

The culture broth then was extracted and the entire sample volume wasspotted in a line onto a 250 mm layer thickness 20 cm×20 cm glass-backedTLC plate containing a fluorescent indicator (254 nm) from Aldrich,Milwaukee, Wis. The doxorubicin was separated from contaminants bychromatography for 2 hours using a mobile phase ofchloroform:methanol:acetic acid:water (80:20:16:6), after which thesilica gel containing the band having R_(f), 0.3-0.4 was scraped fromthe plate. The silica gel was extracted three times with about 1.5 ml ofmethanol each time. The methanol extracts were combined, filteredthrough a 0.2 mm Nylon Acrodisc®13 filter, and air-dried. The driedproduct was resuspended in 500 mL of chloroform:methanol in a ratio of9:1, back-extracted with an equal volume of water that had beenpreviously made alkaline to pH 10.0 using Na₂ CO₃, and the organic phasefrom this extraction procedure was removed and dried. The dried samplewas resuspended in 500 mL of methanol, from which 50 mL were removed forHPLC and TLC analysis. HPLC analysis of this sample confirmed that doxAtransformant cultures converted virtually all of the 500 mg ofdaunomycin to doxorubicin.

The remainder was redried and subjected for mass spectrometry analysis.MS spectra were recorded on a SCIEX API III+ triple quadruple massspectrometer fitted with an atmospheric pressure chemical ionizationsource operating in a positive ion mode. MS spectra were acquired byscanning the first quadruple (Q1), the results are shown in Table 3.

                  TABLE 3    ______________________________________    MS ANALYSIS ON THE DOXORUBICIN PRODUCED ACCORDING    TO EXAMPLE 2                        Calculated                                 Average    Sample              MW       M + 1    ______________________________________    Daunomycin standard 527.51   528.00    Doxorubicin standard                        543.54   544.05    13-Dihydrodaunomycin standard                        529.50   530.01    Doxorubicin from Transformants    Containing doxA     --       544.00    ______________________________________

The results of the MS analysis, shown in Table 3, indicate that thedoxorubicin from doxA transformed cultures has an M+1 of 544.00,essentially the same value as obtained with the doxorubicin standard.The M+1 value of the doxorubicin produced by the doxA transformedculture was not similar to the M+1 values obtained with eitherdaunomycin standard or the 13-dihydrodaunomycin standard.

Example 3

A 50 ml culture of Streptomyces lividans TK24(pANT195) culture wasprepared as in Example 1. This culture was grown for 48 hours at 28° C.and then 25 ml were removed and used to inoculate 200 ml of YEME mediumcontaining 10 mg/L of thiostrepton in a 1.0 L flask, having total, 225ml of culture volume. After incubation for 48 hours at 28° C., 5.0 mg ofdaunomycin-HCl in 1000 ml of distilled water were added to the cultureto give a final concentration of 22.2 mg/ml. A control culture of S.lividans TK24 containing plasmid pANT849 which lacks the doxA gene, wasincubated in the presence of 100 mg of daunomycin-HCl. The cultures wereincubated for 48 hours and then extracted as described in Example 1 andanalyzed by HPLC and TLC. The doxorubicin which migrated in a broad bandhaving an R_(f) of 0.3-0.4 was separated from contaminants bychromatography and prepared for MS analysis as in Example 2. The resultsare shown in Table 4.

The doxA transformed culture converted essentially all of the 5 mg ofdaunomycin to doxorubicin. Notably, the doxA transformed culture wasvirtually dead at the end of 48 hours, whereas the cultures of Examples1 and 2 which received 2 mg/ml of daunomycin were fully viable.Nevertheless, even though the culture was eventually killed by thedaunomycin, the culture converted essentially all of the daunomycin todoxorubicin.

HPLC analysis showed that the doxA transformed cultures convertedgreater than 95% of the daunomycin to doxorubicin. The control cultureconverted essentially 100% of the daunomycin to 13-dihydrodaunomycin,and did not produce doxorubicin.

                  TABLE 4    ______________________________________    MASS SPECTROPHOTOMETRY ANALYSIS ON THE    DOXORUBICIN PRODUCED ACCORDING TO EXAMPLE 3                        Calculated                                 Average    Sample              MW       M + 1    ______________________________________    Doxorubicin Standard                        543.54   543.65    13-Dihydrodaunomycin Standard                        529.50   529.85    Doxorubicin from Transfomants                        --       543.90    Containing doxA    13-Dihydrodaunomycin from Control                        --       529.15    culture    ______________________________________

The results of the MS analysis, shown in Table 4, indicate that thedoxorubicin produced by cultures containing plasmid pANT195, has an M+1of 543.90, essentially the same as obtained with doxorubicin standard.An MS-MS analysis was run on the parent 543.9 peak and is shown in Table5.

                                      TABLE 5    __________________________________________________________________________    MASS SPECTROPHOTOMETRY ANALYSIS OF DOXORUBICIN    PRODUCED ACCORDING TO EXAMPLE 3    Sample        M + 1          Major fragmentation    __________________________________________________________________________    Doxorubicin Standard                  543.65 489.90.sup.m, 396.95, 378.70,                                 360.45 345.95.sup.m 320.85                                 299.45 130.15    Doxorubicin produced according                  543.90 396.80, 378.90, 361.00,    to Example 3    13-Dihydrodaunomycin                  529.85 382.85, 365.30, 346.35,                                 320.85, 129.35, 113.10    Standard    Control       529.15 497.20.sup.m, 482.00.sup.m, 382.80,                                 364.60, 320.75, 305.75,    culture extract              129.95    __________________________________________________________________________     .sup.1 The product sample was significantly less concentrated than the     standard sample, leading to recovery of only the most abundant     fragmentation species.     .sup.mminor fragmentation species.

The MS-MS analysis on 543.90 peak from the doxorubicin produced inExample 3 shows daughter peaks which are essentially identical to thedaughter peaks from the doxorubicin standard. Thus, the doxorubicinproduced by the transformants containing doxA has the M+1 and MS/MSfragmentation patterns of standard doxorubicin. Similarly, the cultureextract from the control culture had an M+1 and fragmentation patternsimilar to that of standard 13-dihydrodaunomycin.

Example 4

Fifty ml cultures of Streptomyces lividans TK24(pANT195) and S. lividansTK24(pANT849) as a control were inoculated and prepared as in Example 1except that they were grown at 28° C. for 48 hours. At that time, 100 mgof daunomycin in 10 mL of distilled water was added to the cultures fora final concentration of 2 mg/ml. The cultures were further incubatedfor 24 hours. The cultures were then extracted as described in Example 1and subjected to HPLC analysis.

The transformed culture containing the doxA gene converted greater than95% of the daunomycin to doxorubicin within 24 hours. The controlculture converted approximately 100% of the daunomycin to13-dihydrodaunomycin.

Example 5

The procedure of Example 4 was repeated except that 100 mg of13-dihydrodaunomycin, rather than daunomycin, were added to the culturesand the cultures were further incubated for 48 hours rather than 24hours.

In 48 hours, the culture containing plasmid doxA (pANT195) converted100% of the 13-dihydrodaunomycin to doxorubicin. The control culture didnot convert the 13-dihydrodaunomycin.

Example 6

Fifty ml cultures of Streptomyces lividans TK24(pANT 196) and S.lividans TK24(pIJ702) as a control, were treated as in Example 4 exceptthat the cultures were incubated with daunomycin for 72 hours.

The culture containing plasmid pANT196, which contains the doxA geneexpressed from the melC1 promoter, converted 20% of the daunomycin todoxorubicin. Thus the melC1 promoter is less preferred than the snpApromoter. The control cultures converted 100% of the daunomycin to13-dihydrodaunomycin.

Example 7

50 ml cultures of Streptomyces lividans TK24(pANT192), having wild typeand snpA promoters, Streptomyces lividans TK24(pANT193), having wildtype and snpA promoters, Streptomyces lividans TK24(pANT194) which lacksthe snpA-promoter and snpR activator gene, and S. lividans TK24(pANT849)as a control, were prepared and analyzed prepared as in Example 4,except that the cultures were incubated in the presence of thedaunomycin for 48 hours. The results are presented in Table 6.

                  TABLE 6    ______________________________________    COMPARISON OF DIFFERENT PLASMIDS ON    PERCENT CONVERSION OF DAUNOMYCIN TO DOXORUBICIN    Plasmid       Products    ______________________________________    pANT192       75% 13-dihydrodaunomycin/25%    pANT193       80% doxorubicin/20% 13-    pANT194       90% 13-dihydrodaunomycin/                  10% doxorubicin    pANT195       100% doxorubicin    pANT849       100% 13-DHD    (control)    ______________________________________

As shown in Table 6, the culture containing pANT192, which containsdoxA, converted 25% of daunomycin to doxorubicin and 75% of daunomycinto the 13-dihydrodaunomycin. The culture containing plasmid pANT193,which contains doxA, converted 80% of daunomycin to doxorubicin and 20%of daunomycin to the 13-dihydrodaunomycin. The culture containingplasmid pANT194, which contains doxA but lacks the snpA-promoter andsnpR activator, converted 10% of daunomycin to doxorubicin and 90% ofdaunomycin to the 13-dihydrodaunomycin. The control culture convertedthe daunomycin to 13-dihydrodaunomycin, but not doxorubicin.

Example 8

Fifty ml cultures of Streptomyces lividans TK24(pANT195) and S. lividansTK24(pANT849) as a control, were prepared as in Example 4, except thatthe pH of the YEME medium was adjusted before inoculation using NaOH orHCl to provide an initial culture pH as shown in Table 7. The cultureswere further incubated for 48 hours, rather than 24 hours. The resultsare shown in Table 7.

                  TABLE 7    ______________________________________    EFFECT OF CULTURE PH ON DOXORUBICIN PRODUCTION                        Percent Daunomycin    Initial pH Final pH bioconverted to Doxorubicin    ______________________________________    6.0        7.0       50%    6.5        7.6       90%    7.0        7.9      100%    7.5        7.0      100%    8.0        --       No growth or                        bioconversion    ______________________________________     The % conversion is approximate.

The culture containing plasmid pANT195 which contains doxA, and whichwas initially at pH of 7.0 or 7.5, converted 100% of the daunomycin todoxorubicin. The cultures containing plasmid doxA which were initiallyat pH 6.0 converted 50% of the daunomycin to doxorubicin. The culturescontaining plasmid doxA which were initially at pH 6.5 converted 90% ofthe daunomycin to doxorubicn. Accordingly it is preferred that thetransformed host cultures be grown at an initial pH of higher than 6.5.

Example 9

The procedure of Example 4 was repeated except that cultures were grownat either 22° C., 28° C., or 37° C. and incubated with the daunomycinfor 48 hours at such temperatures.

All three of the cultures containing plasmid doxA converted 100% of thedaunomycin to doxorubicin.

Example 10

A 50 ml culture of Streptomyces lividans TK24(pANT195), inoculated andprepared as in Example 1, was grown at 28° C. for 48 hours. At thattime, the cultures were harvested by centrifugation at 10,000'g in ahigh speed centrifuge, washed once with 100 mM3-(N-morpholino)propanesulfonic acid buffer at pH 7.2. The cells fromthe cultures were reconstituted in 5.0 ml of the 100 mM3-(N-morpholino)propanesulfonic acid buffer to give a final volume of6.0 ml which included the volume of the packed cell mass, resulting inan approximately 8-fold concentration of the recombinant mycelia inbuffer. Then 100 mg of daunomycin were added in 10 mL of distilled waterfor a final concentration of 16.7 mg/ml of daunomycin. A concentrationof 16.7 mg/ml of daunomycin is toxic to the host. The culture wasfurther incubated for 7.0 hours, after which it was extracted asdescribed in Example 1 and subjected to HPLC analysis.

In 7 hours, the concentrated cultures containing the plasmid with a doxAinsert converted about 25% of the daunomycin to doxorubicin.

Example 11

Five ml each of APM seed medium containing 10 mg/ml of thiostrepton wereinoculated by loop from R2YE agar plates, containing 50 mg/mlthiostrepton of S. peucetius 29050(pANT195) and S. peucetius29050(pANT849). Each culture was grown at 28° C. for 48 hours. Fifty mleach of GPS "production" medium containing 10 mg/ml of thiostrepton wereinoculated with 2.5 mls of seed culture grown in APM seed medium. Thecultures were grown at 28° C. for 48 hours as in Example 1. Then 100 mgof daunomycin in 10 mL of distilled water for a final concentration of 2mg/ml of daunomycin was added to the cultures. The cultures were furtherincubated for 48 hours, then extracted as described in Example 1.

After 48 hours, the culture containing plasmid pANT195, which containsthe doxA gene, converted about one-half of the daunomycin todoxorubicin. The control cultures did not convert daunomycin todoxorubicin.

Example 12

Fifty ml cultures of Streptomyces coelicolor CH999 (pANT195), andStreptomyces coelicolor CH999 (pANT849) as a control, were used in theprocedure of Example 4 except that the cultures were incubated with thedaunomycin for 48 hours.

Again the cultures containing pANT195 converted 100% of the daunomycinto doxorubicin, while the control converted 80% of the daunomycin to13-dihydrodaunomycin. In the control cultures 20% of the daunomycin wasnot converted.

Example 13

The procedure of Example 12 was repeated, except that after thedaunomycin addition, the cultures were only incubated for 1, 2, or 4hours.

After 1 hour, 0.7% of the daunomycin was converted to doxorubicin by thecultures which contained pANT195. After 2 hours, 1.0% of the daunomycinwas converted and by 4 hours 15% of the daunomycin was converted todoxorubicin. The control cultures which lacked the plasmid containingdoxA did not convert any of the daunomycin.

Example 14

50 ml cultures of Streptomyces sp. strain C5(pANT195) and Streptomycessp. strain C5(pANT849) as a control, were grown at 28° C. for 72 hoursin NDYE medium as described in Example 1. After 72 hours, 100 mg ofunlabelled daunomycin and 5 mCi of ³ H-daunomycin, having a specificradioactivity of 5.0 Ci/mmol, were added to each culture and they wereincubated for another 48 hours. The cultures were extracted as describedin Example 1 and analyzed by TLC and autoradiography.

The cultures of containing plasmid pANT195 converted approximately 5% ofthe radiolabelled daunomycin to doxorubicin. No other products otherthan the substrate daunomycin and doxorubicin were observed in thesecultures. The control cultures converted approximately 90% of theradiolabelled daunomycin to baumycin A1 and baumycin A2.

Example 15

50 ml cultures of Streptomyces sp. strain C5, which does not appear tosynthesize doxorubicin, and the following mutants of Streptomyces sp.strain C5: SC5-dauA74, SC5-dauCE147, SC5-dauE24, and SC5-dauH54, weregrown for 48 hours in NDYE medium. These mutants do not synthesizedaunomycin. At 48 hours, 100 mg of daunomycin was added for a finalconcentration of 2 mg/ml, and the cultures were incubated for 36 hours.The products were extracted as in Example 1 and subjected to HPLCanalysis.

All Streptomyces sp. strain C5 cultures, each of which lacked a plasmidcontaining doxA, converted about 10% of the daunomycin to doxorubicin.Baumycins A1 and A2 were also detected.

Example 16

The procedure of Example 4 was repeated except that the cultures werethen incubated for 48 hours with 100 mg, for a final concentration of2ug/ml of one of the following: carminomycin, idarubicin, daunomycinone,or carminomycinone.

Incubation of S. lividans TK24(pANT195) cultures with carminomycin oridarubicin, resulted in greater than 85% recovery of carminomycin andidarubicin. The cultures which contained plasmid pANT195 converted 100%of the daunomycinone to 13-hydroxydaunomycinone and 100% of thecarminomycinone to 13-hydroxycarminomycinone. The control cultures,which lack the doxA gene, converted 100% of the carminomycin,idarubicin, daunomycinone, and carminomycinone to their 13-dihydroderivatives.

Example 17

The procedure of Example 4 was repeated except that the culturesreceived 100 mg of 13-dihydrocarminomycin rather than daunomycin. Thecultures were then incubated for 36 hours and analyzed by TLC and HPLC.

The cultures containing plasmid pANT195 converted 100% of the13-dihydrocarminomycin to carminomycin. No other products were observed.In the control culture, none of the 13-dihydrocarminomycin wasconverted. The doxA gene confers the ability to oxidize the C-13hydroxyl function of the 13-dihydro-carminomycin to a keto function.

Novel Synthesis of 13-deoxycarminomycin and 13-deoxydaunomycin

Example 18

e-Rhodomycin D compound was converted to both 13-deoxycarminomycin and13-deoxydaunomycin, by host microorganisms containing plasmid whichcontains the Streptomyces sp. strain C5 dauP gene which encodese-rhodomycin D esterase and the Streptomyces sp. strain C5 dauK genewhich encodes carminomycin 4-O-methyltransferase.

Plasmid pANT144 as shown in FIG. 12, and described in Dickens, M. L.,et. al., (1995) "Analysis of Clustered Genes Encoding both Early andLate Steps in Daunomycin Biosynthesis by Streptomyces sp. strain C5 " J.Bacteriol. . volume 177, pages 536-543, was introduced into S. lividansTK24 by protoplast transformation. S. lividans TK24(pANT144) was grownfor 48 hours in 50 ml of YEME medium containing 10 mg/ml of thiostreptonand then used to inoculate 450 ml of the YEME medium which contained 10mg/ml of thiostrepton for a total culture volume of 500 ml, in a twoliter flask. The resultant 500 ml culture was incubated for 48 hours at28° C. as in Example 1. Next, 5.0 mg of e-rhodomycin D, the glycone ofe-rhodomycinone, from the National Cancer Institute, Drug Synthesis andChemistry Branch, Bethesda, Md. designated compound #263854-H, wereadded to the culture for a final concentration of 10 mg/ml and thecultures were incubated for an additional 48 hours. The culture then wasadjusted to pH 8.5, and then extracted twice, each with 1 volume ofchloroform:methanol (9:1) as in Example 3. The organic extract wasreduced to dryness, reconstituted in 500 mL of chloroform:methanol(9:1), filtered, back extracted, dried, and reconstituted in 2.0 ml ofmethanol. The extract was separated and extracted as in Example 2. R_(f)values for 13-deoxycarminomycin and 13-deoxydaunomycin wereapproximately 0.60 and 0.64, respectively. The 13-deoxycarminomycin and13-deoxydaunomycin were reduced to dryness and each was brought up againin 50 ml of methanol.

Methods of Producing Anthracyclines Employing Daunomycin C-14Hydroxylase

Example 19

904 ml of the daunomycin C-14 hydroxylase produced according to themethod of Example B, containing approximately 1 mg of total protein, wasincubated in a 16 mm well of a 24 well culture plate at 30° C. withshaking for 2 hours with 25 mg in 5 ml of either daunomycin,13-dihydrodaunomycin, or 13-dihydrocarminomycin. The final volume ofeach well was 1.0 ml; 0.1M sodium phosphate buffer at pH 7.5, was addedto bring the total volume to 1 ml, as needed. After 2 hours ofincubation, the pH of each reaction mixture was increased to pH 8.5using 1 M NaOH and each was extracted twice each with 500 ml ofchloroform: methanol (9:1). The organic layers were combined, reduced todryness, reconstituted in 10 ml of methanol, separated and analyzed byTLC as in Example 1.

The daunomycin C-14 hydroxylase converted 50% of the13-dihydrocarminomycin to carminomycin and 50% of the13-dihydrodaunomycin to daunomycin.

Example 20

The procedure of example 19 was repeated except that 10 ml of NADH, 1 mMfinal concentration; 10 ml of NADPH, 1 mM final concentration, wereadded.

The daunomycin C-14 hydroxylase converted 100% of the13-dihydrocarminomycin to carminomycin and 100% of the13-dihydrodaunomycin to daunomycin.

Example 21

The procedure of Example 20 was repeated except that the followingreagents, available from Sigma Chemical Co., were added: 20 ml ofglucose-6-phosphate; 10 mM final concentration; 10 ml of NADP⁺, 1 mMfinal concentration; 1.0 ml of glucose-6-phosphate dehydrogenase; 0.84units, final activity; 20 ml of spinach ferredoxin, 44 mg finalconcentration; and 10 ml of spinach ferredoxin-NADP⁺ reductase, 0.05units final activity. The glucose-6-phosphate, glucose-6-phosphatedehydrogenase and NADP⁺ constitute a "NADPH-regenerating system". After2 hours of incubation, the extracts were extracted and analyzed by TLCand HPLC.

The daunomycin C-14 hydroxylase converted 100% of the13-dihydrocarminomycin to carminomycin and 100% of the13-dihydrodaunomycin to daunomycin, as shown by TLC. HPLC revealed theconversion of about 5% of the daunomycin to doxorubicin.

Example 22

The procedure of Example 21 was repeated, except 10 ml of flavin adeninemononucleotide from Sigma, 10 mg final concentration; and 10 mg in 10ml, flavin adenine dinucleotide, were also added.

100% of the 13-dihydrocarminomycin was converted to carminomycin and100% of the 13-dihydrodaunomycin was converted to daunomycin by thedaunomycin C-14 hydroxylase. Doxorubicin was not detected using TLC.

Example 23

The procedure of Example 20 was repeated except that differentanthracyclines were used, incubation was for 1 hour and 10 ml of NADP⁺,1 mM final concentration was added. 25 mg in 5 ml the followinganthracyclines were used: either e-rhodomycin D, 13-deoxydaunomycin fromexample 18, or 13-deoxycarminomycin, from example 18.

About 20% of the 13-deoxycarminomycin was converted to13-dihydrocarminomycin and about 80% was converted to carminomycin; and13-deoxydaunomycin was converted to about 20% 13-dihydrodaunomycin andabout 80% daunomycin. The e-rhodomycin D did not appear to be converted.

Thus, the daunomycin C-14 hydroxylase converted 13-deoxycarminomycin to13-dihydrocarminomycin and carmninomycin; 13-dihydrocarminomycin tocarminomycin; 13-deoxydaunomycin to 13-dihyrodaunomycin and daunomycin;and 1 3-dihydrodaunomycin to daunomycin. Thus, daunomycin C-14hydroxylase catalyzes the oxidation of the C-13 methylene to a C-13hydroxyl function, and catalyzes the oxidation of the C-13 hydroxylfunction to C-13 keto function. The daunomycin C-14 hydroxylase isuseful for making 13-dihydrocarminomycin, carminomycin,13-dihydrodaunomycin and daunomycin.

Example 24

The procedure of Example 22 was repeated except that the daunomycin C-14hydroxylase was incubated with daunomycin for 18 hours rather than 2hours and in 25 ml erylenmyer flasks shaken at 250 rpm on a rotaryshaker.

Doxorubicin was not detected by HPLC or TLC.

Example 25

The procedure of Example 24 was repeated except that the reagent volumeswere tripled. The reagent concentrations however were not increased.

Approximately 5% of the daunomycin was converted to doxorubicin asdetermined by a HPLC.

Example 26

The procedure of Example 24 was repeated except that the reagent volumeswere quintupled. The reagent concentrations however, were not increased.

Approximately 20 to 25% of the daunomycin was converted to doxorubicin,as determined by HPLC.

The present invention includes: the DNA sequences encoding a proteindaunomycin C-14 hydroxylase, which adds a hydroxyl group to carbon 14 ofdaunomycin; the messenger RNA transcript of such DNA sequence; and anisolated protein which adds a hydroxyl group to carbon 14 of daunomycin.

For example, the DNA sequences include: DNA molecules which, but for thedegeneracy of the genetic code would hybridize to DNA encoding thedaunomycin C-14 hydroxylase, thus the degenerate DNA which encodes thedaunomycin C-14 hydroxylase protein; DNA strands complementary to: DNAsequences encoding the daunomycin C-14 hydroxylase protein including DNAin FIGS. 3, 6, 9 and 11; heterologous DNA having substantial sequencehomology to the DNA encoding the daunomycin C-14 hydroxylase protein,including the DNA sequences in FIGS. 3, 6, 9 and 11 or portions thereof.

The daunomycin C-14 hydroxylase protein includes, for example, thedaunomycin C-14 hydroxylase protein of strains other than Streptomycessp. strain C5; proteins having 75% homology to the proteins in FIGS. 3,6, 9 and 11, and proteins or portions thereof having substantially thesame amino acid sequence as shown in FIGS. 3, 6, 9 and 11.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 9    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #                24GGTG CCTC    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    # 20               GGGG    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 48 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #                48CTAG AGCTCAAGCT TTAAACTAGT TAACGCGT    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 3196 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: mat.sub.-- - #peptide              (B) LOCATION: 1498..2764    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1498..2764    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - GGTACCCGCG CATCGATGTC ATGGCCGGCA ACGCCGGCGG CATGTTCTGG TC - #GCGCACCA      60    - CGACCCAGGA CGGGTTCGAG GCCACCCTCC AGGTCAATCA TCTCGCGGGC TT - #CCTGCTGG     120    - CACGGCTGCT GCGGGAGCGG CTCGCGGGCG GGCGGTTGAT CCTCACCTCG TC - #CGACGCGT     180    - ACACCCAGGG CCGGATCGAC CCGGACGATC TCAACGGCGA CCGTCACCGC TA - #CAGCGCGG     240    - GCCAGGCGTA CGGCACGTCC AAACAGGCCA ACATCATGAC CGCCACGGAG GC - #CGCCCGGC     300    - GCTGGCCGGA CGTGCTGACG GTCAGCTACC ACCCCGGCGA GGTCCGCACC CG - #CATCGGGC     360    - GGGGCACAGT CGCCTCGACC TACTTCCGGT TCAACCCCTT CCTGCGGTCC GC - #GGCCAAGG     420    - GCGCCGACAC TCTCGTGTGG CTGGCGGCCG CGCCGGCCGA GGAGTTGACC AC - #GGGCGGCT     480    - ACTACAGCGA CCGGCGGCTG TCCCCGGTGA GCGGCCCGAC CGCCGACGCC GG - #CCTCGCGG     540    - CCAAGCTCTG GGAGGCCAGC GCGGCCGCCG TCGGCCACAC CGCGCGCTGA CC - #GCGGCGGG     600    - CCTCCCCGCC CGCATGCCCG TCTCATCCGC GAGCGCAGAC GCTCGTGTGC CG - #ATCCGTCG     660    - AAAGGAACGA TTCGTGACCA GGTTCGCGCC CGGCGCCCCC GCATGGTTCG AC - #CTCGGGTC     720    - GCCCGATGTC GCCGCCTCGG CCGACTTCTA CACCGGCCTC TTCGCGTGGA CC - #GCGACCGT     780    - GGTCAGCGAC CCGGGTGCCG GGGGATACAC TACTTTCAGC TCCGACGGGA AG - #CCTGTCGC     840    - CGCGGTCGCC CGCCATCAGA TCGACACGCC CTACCACCGT CCGTACGGGC CC - #GGCAAGCA     900    - CCAGCACGGC ATGCCGGCCA TCTGGACCGT GTACTTCGCC ACCAACGACG CC - #GACGCACT     960    - GACCAAACGG GTCGAAGCGG CGGGTGGCGA CGTCATCATG CACCCGATGG AC - #GTCCTCGG    1020    - TCTCGGCCGG ATGGCGGTCT TCGCCGACCC ATCGGGGGCC GCGTTCGCGG TG - #TGGCGCAA    1080    - GGGCGTCATG GAGGGCGCGG AGGTGACGGG CGTGCCCGGC TCGGTCGGCT GG - #GTGGAACT    1140    - GGTGACCGAC GACATCGGGA CCGCCCGTGG CTTCTACCGT GCGACCCTCG GC - #CTGGCTCC    1200    - GGCCGACACC GGACGCAAGG GCGTCACCGA CCCGGTTTGG CACATCCATG AC - #ACACCGGT    1260    - CGCCGGCACC CGGGAACTGG GCACGACCGG CGCGGTACGG CCCCACTGGG CC - #GTGCTGTT    1320    - CTCCGTGCAC GACTGCGACG CGACGGTCCG GCGGGCCGTC GAACTCGGCG GC - #TCCGTCGA    1380    - GAACGAGCCC GTCGACACCC CCAGGGGGCG GCGGGCGGAC CTGCTCGACC CG - #CACGGGGC    1440    - CGGCTTCTCG GTGGTCGAAC TGCGGGAGGC GTACCCCGCG GCGGCGGACG GT - #GCCTC    1497    - ATG AGC GGC GAG GCG CCG CGG GTG GCC GTC GA - #C CCG TTC TCG TGT CCC    1545    Met Ser Gly Glu Ala Pro Arg Val Ala Val As - #p Pro Phe Ser Cys Pro    #                 15    - ATG ATG ACC ATG CAG CGC AAA CCC GAG GTG CA - #C GAC GCA TTC CGA GAG    1593    Met Met Thr Met Gln Arg Lys Pro Glu Val Hi - #s Asp Ala Phe Arg Glu    #             30    - GCG GGC CCC GTC GTC GAG GTG AAC GCC CCC GC - #G GGC GGA CCC GCC TGG    1641    Ala Gly Pro Val Val Glu Val Asn Ala Pro Al - #a Gly Gly Pro Ala Trp    #         45    - GTC ATC ACC GAT GAC GCC CTC GCC CGC GAG GT - #G CTG GCC GAT CCC CGG    1689    Val Ile Thr Asp Asp Ala Leu Ala Arg Glu Va - #l Leu Ala Asp Pro Arg    #     60    - TTC GTG AAG GGA CCC GAT CTC GCG CCC ACC GC - #C TGG CGG GGG GTG GAC    1737    Phe Val Lys Gly Pro Asp Leu Ala Pro Thr Al - #a Trp Arg Gly Val Asp    # 80    - GAC GGT CTC GAC ATC CCC GTT CCG GAG CTG CG - #T CCG TTC ACG CTC ATC    1785    Asp Gly Leu Asp Ile Pro Val Pro Glu Leu Ar - #g Pro Phe Thr Leu Ile    #                 95    - GCC GTG GAC GGT GAG GAC CAC CGG CGT CTG CG - #C CGC ATC CAC GCA CCG    1833    Ala Val Asp Gly Glu Asp His Arg Arg Leu Ar - #g Arg Ile His Ala Pro    #           110    - GCG TTC AAC CCG CGC CGG CTG GCC GAG CGG AC - #G GAT CGC ATC GCC GCC    1881    Ala Phe Asn Pro Arg Arg Leu Ala Glu Arg Th - #r Asp Arg Ile Ala Ala    #       125    - ATC GCC GAC CGG CTG CTC ACC GAA CTC GCC GA - #C TCC TCC GAC CGG TCG    1929    Ile Ala Asp Arg Leu Leu Thr Glu Leu Ala As - #p Ser Ser Asp Arg Ser    #   140    - GGC GAA CCG GCC GAG CTG ATC GGC GGC TTC GC - #G TAC CAC TTC CCG CTG    1977    Gly Glu Pro Ala Glu Leu Ile Gly Gly Phe Al - #a Tyr His Phe Pro Leu    145                 1 - #50                 1 - #55                 1 -    #60    - TTG GTC ATC TGC GAA CTG CTC GGC GTG CCG GT - #C ACC GAT CCG GCA ATG    2025    Leu Val Ile Cys Glu Leu Leu Gly Val Pro Va - #l Thr Asp Pro Ala Met    #               175    - GCC CGC GAG GCC GTC GGC GTG CTC AAG GCA CT - #C GGC CTC GGC GGC CCG    2073    Ala Arg Glu Ala Val Gly Val Leu Lys Ala Le - #u Gly Leu Gly Gly Pro    #           190    - CAG AGC GCC GGC GGT GAC GGC ACG GAC CCT GC - #C GGG GAC GTG CCG GAC    2121    Gln Ser Ala Gly Gly Asp Gly Thr Asp Pro Al - #a Gly Asp Val Pro Asp    #       205    - ACG TCG GCG CTG GAG AGC CTT CTC CTC GAA GC - #C GTG CAC GCG GCC CGG    2169    Thr Ser Ala Leu Glu Ser Leu Leu Leu Glu Al - #a Val His Ala Ala Arg    #   220    - CGG AAA GAC ACC CGG ACC ATG ACC CGC GTG CT - #C TAT GAA CGC GCA CAG    2217    Arg Lys Asp Thr Arg Thr Met Thr Arg Val Le - #u Tyr Glu Arg Ala Gln    225                 2 - #30                 2 - #35                 2 -    #40    - GCA GAG TTC GGC TCG GTC TCC GAC GAC CAG CT - #C GTC TAC ATG ATC ACC    2265    Ala Glu Phe Gly Ser Val Ser Asp Asp Gln Le - #u Val Tyr Met Ile Thr    #               255    - GGA CTC ATC TTC GCC GGC CAC GAC ACC ACC GG - #C TCG TTC CTG GGC TTC    2313    Gly Leu Ile Phe Ala Gly His Asp Thr Thr Gl - #y Ser Phe Leu Gly Phe    #           270    - CTG CTT GCG GAG GTC CTG GCG GGC CGT CTC GC - #G GCG GAC GCC GAC GGG    2361    Leu Leu Ala Glu Val Leu Ala Gly Arg Leu Al - #a Ala Asp Ala Asp Gly    #       285    - GAC GCC ATC TCC CGG TTC GTG GAG GAG GCG CT - #G CGC CAC CAC CCG CCG    2409    Asp Ala Ile Ser Arg Phe Val Glu Glu Ala Le - #u Arg His His Pro Pro    #   300    - GTG CCC TAC TCG TTG TGG AGG TTC GCT GCC AC - #G GAG GTG GTC ATC CGC    2457    Val Pro Tyr Ser Leu Trp Arg Phe Ala Ala Th - #r Glu Val Val Ile Arg    305                 3 - #10                 3 - #15                 3 -    #20    - GGT GTC CGG CTG CCC CGC GGA GCG CCG GTA CT - #G GTG GAC ATC GAG GGC    2505    Gly Val Arg Leu Pro Arg Gly Ala Pro Val Le - #u Val Asp Ile Glu Gly    #               335    - ACC AAC ACC GAC GGC CGC CAT CAC GAC GCC CC - #G CAC GCT TTC CAC CCG    2553    Thr Asn Thr Asp Gly Arg His His Asp Ala Pr - #o His Ala Phe His Pro    #           350    - GAC CGC CCT TCG AGG CGG CGG CTC ACC TTC GG - #C GAC GGG CCG CAC TAC    2601    Asp Arg Pro Ser Arg Arg Arg Leu Thr Phe Gl - #y Asp Gly Pro His Tyr    #       365    - TGC ATC GGG GAG CAG CTC GCC CAG CTG GAA TC - #G CGC ACG ATG ATC GGC    2649    Cys Ile Gly Glu Gln Leu Ala Gln Leu Glu Se - #r Arg Thr Met Ile Gly    #   380    - GTA CTG CGC AGC AGG TTC CCC CAA GCC CGA CT - #G GCC GTG CCG TAC GAG    2697    Val Leu Arg Ser Arg Phe Pro Gln Ala Arg Le - #u Ala Val Pro Tyr Glu    385                 3 - #90                 3 - #95                 4 -    #00    - GAG TTG CGG TGG TGC AGG AAG GGG GCC CAG AC - #A GCG CGG CTC ACT GAC    2745    Glu Leu Arg Trp Cys Arg Lys Gly Ala Gln Th - #r Ala Arg Leu Thr Asp    #               415    #GCACGGGACC          2794 T GATGGGCCGA CCGCGACCCG    Leu Pro Val Trp Leu Arg                420    - GCCCACCGCC CATCGCGCGG TGGGCGGTCC CGTGCCGGTC GCCCGGTGCG GT - #CCTCTCCC    2854    - GACGCTCGCT CCCCCTGTGA CTTTCTCACA TCGAGACGTG ACGAAATAAT CC - #CAGCAAGT    2914    - GCCATGCACA CTTTCATGGC GGACATTCAC TTGCGAGGAT GGAGTGAGCA CA - #CGGGGCCG    2974    - CCCGAGACAC CCTACGGCCG CCGGAAGTAT GCCACCTGTT GACGCGAATG GA - #ACGCCACA    3034    - GAGGGAGCAC CGGCAATGCA GATCAATATG TTGGGCCCGC TCGTTGCACA TC - #ACAATGGC    3094    - ACGTCGGTGA CCCCGATAGC CAGAAAACCC CGGCAGGTAT TCTCACTGCT CG - #CTCTTCAG    3154    #3196              CGGT CCCCGCGCTG ATGGAGGAGC TC    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 422 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Met Ser Gly Glu Ala Pro Arg Val Ala Val As - #p Pro Phe Ser Cys Pro    #                 15    - Met Met Thr Met Gln Arg Lys Pro Glu Val Hi - #s Asp Ala Phe Arg Glu    #             30    - Ala Gly Pro Val Val Glu Val Asn Ala Pro Al - #a Gly Gly Pro Ala Trp    #         45    - Val Ile Thr Asp Asp Ala Leu Ala Arg Glu Va - #l Leu Ala Asp Pro Arg    #     60    - Phe Val Lys Gly Pro Asp Leu Ala Pro Thr Al - #a Trp Arg Gly Val Asp    # 80    - Asp Gly Leu Asp Ile Pro Val Pro Glu Leu Ar - #g Pro Phe Thr Leu Ile    #                 95    - Ala Val Asp Gly Glu Asp His Arg Arg Leu Ar - #g Arg Ile His Ala Pro    #           110    - Ala Phe Asn Pro Arg Arg Leu Ala Glu Arg Th - #r Asp Arg Ile Ala Ala    #       125    - Ile Ala Asp Arg Leu Leu Thr Glu Leu Ala As - #p Ser Ser Asp Arg Ser    #   140    - Gly Glu Pro Ala Glu Leu Ile Gly Gly Phe Al - #a Tyr His Phe Pro Leu    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Val Ile Cys Glu Leu Leu Gly Val Pro Va - #l Thr Asp Pro Ala Met    #               175    - Ala Arg Glu Ala Val Gly Val Leu Lys Ala Le - #u Gly Leu Gly Gly Pro    #           190    - Gln Ser Ala Gly Gly Asp Gly Thr Asp Pro Al - #a Gly Asp Val Pro Asp    #       205    - Thr Ser Ala Leu Glu Ser Leu Leu Leu Glu Al - #a Val His Ala Ala Arg    #   220    - Arg Lys Asp Thr Arg Thr Met Thr Arg Val Le - #u Tyr Glu Arg Ala Gln    225                 2 - #30                 2 - #35                 2 -    #40    - Ala Glu Phe Gly Ser Val Ser Asp Asp Gln Le - #u Val Tyr Met Ile Thr    #               255    - Gly Leu Ile Phe Ala Gly His Asp Thr Thr Gl - #y Ser Phe Leu Gly Phe    #           270    - Leu Leu Ala Glu Val Leu Ala Gly Arg Leu Al - #a Ala Asp Ala Asp Gly    #       285    - Asp Ala Ile Ser Arg Phe Val Glu Glu Ala Le - #u Arg His His Pro Pro    #   300    - Val Pro Tyr Ser Leu Trp Arg Phe Ala Ala Th - #r Glu Val Val Ile Arg    305                 3 - #10                 3 - #15                 3 -    #20    - Gly Val Arg Leu Pro Arg Gly Ala Pro Val Le - #u Val Asp Ile Glu Gly    #               335    - Thr Asn Thr Asp Gly Arg His His Asp Ala Pr - #o His Ala Phe His Pro    #           350    - Asp Arg Pro Ser Arg Arg Arg Leu Thr Phe Gl - #y Asp Gly Pro His Tyr    #       365    - Cys Ile Gly Glu Gln Leu Ala Gln Leu Glu Se - #r Arg Thr Met Ile Gly    #   380    - Val Leu Arg Ser Arg Phe Pro Gln Ala Arg Le - #u Ala Val Pro Tyr Glu    385                 3 - #90                 3 - #95                 4 -    #00    - Glu Leu Arg Trp Cys Arg Lys Gly Ala Gln Th - #r Ala Arg Leu Thr Asp    #               415    - Leu Pro Val Trp Leu Arg                420    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 3013 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - GCAGGCGGTA CCGCCGACCC GCTGCATCCC CCGCACCGCC GTCCCCCCCC AG - #GGCATCTC      60    - CCGTCGGGTT ACGGGAAGGG GGCCGGGGTA CCCGGTCGTC ACGGGAGGGC TG - #GGACGAGT     120    - GCCCCCGACC CACTGCGTTC CAGCCACTCC CGGTACGCCG GGGCCTGCCG GG - #CGACCTCC     180    - CCGTAGGCCT CCGCGAGGTC GGGGTAGACG CCCTCCAGTT CCGTGTCCGT GC - #GGGCCGCC     240    - AGCAGCAGCC GTACGCCGAG CGGGTCGCCG TGCAGCCGGC GGACGGCCGT CT - #CGGCGCGG     300    - GAGGGCGAGG TCGGCTGGAC CACGGTGACG ACCTCGCCGG TGGCGACCAG GT - #ACGCGGCG     360    - GAGTGGTAGT CCCCGTGCAG GATGCGCGAG TCGAGTCCCT CGGCGCGCAG GA - #CCCGGCGC     420    - ACCGCGTTCC ACTCGCCGTC GACGGTGGGG TCGATCATCC AGCGGGTCGT GG - #GCCAGGTC     480    - GGCGAGGCGT ACGACGTGGC TTCGGCGGCC GGGTGGTCGG CCGGCAGGCA GA - #CGAACTGC     540    - GGTTCCCGCT GGACCAGTAC GCGGACCCGG AGCCCTTCGG GGACGCGCAG GC - #TGCCCTCG     600    - ACCTCGTGCA CGAAGGCGAC GTCGAGGTGG CCGTCGGCCA CCATGCGCAG CA - #GGGCGTTG     660    - GCGGAGACGT CCATGTGCAG GGTGGGTTCC TGCCAGTGCC GGAGCCGGCG CA - #GCCAGCCC     720    - GCCAGGGCCC GGCTGGCCGT GGAGCCGACG CGCAGGCTGG CGTCCGCGAC GG - #CGGCGGCG     780    - CGGGCCTCGC TGACGAGGGA GCACAATTCG GCCACCAGGG GGCGGGCACG AC - #TGAGAACC     840    - AGCCGGCCCA GCGGTGTGGG GCGGCAGCCG GTGCGGGCCC GGACGAACAG GG - #CACGGCCC     900    - AGCTCGTGTT GGATGCGCCG CAGCTGCGTG CTCAACGAGG GCTGTGTCAC TC - #CCAGTTGG     960    - CGTGCCGCGC GGTGCAGGCT GCCGGTGTCG GCGATGGCGC ACAGCGCCCT GA - #GGTGCCTG    1020    - ACCTCAAGCT CCATGTCCTG GGAGGGTAAG GCGGAAGTTC AGCTTTCACC AG - #ACATACAA    1080    - AATGGCGACC GATCAGGACC ATCGGGCCTT CACGGCGCGA GGCGTCGGCC CG - #GATCGGCA    1140    - GGGGCCCCGG CCGGGGCCGC CGGGCAGGGC GGCGCAGGTG GGGACGGAGG GG - #GATAGGGC    1200    - GGCCCTATCG GCGGTTGCCA TCATCACAAC GGCCGTACGG GCACGGACAC TC - #ACGATGTC    1260    - TGACTCATCC CCCCACCTCG AGGAGTCATC GATGCGCATG CGGAGGGGTG CC - #TCATCAGC    1320    - GGCCCTATCG GCGGTTGCCA TCATCACAAC GGCCGTACGG GCACGGACAC TC - #ACGATGTC    1380    - TGACTCATCC CCCCACCTCG AGGAGTCATC GATGCGCATG CGGAGGGGTG CC - #TCATGAGC    1440    - GCGGGCGGAC CCGCCTGGGT CATCACCGAT GACGCCCTCG CCCGCGAGGT GC - #TGGCCGAT    1500    - CCCCGGTTCG TGAAGGACCC CGATCTCGCG CCCACCGCCT GGCGGGGGGT GG - #ACGACGGT    1560    - CTCGACATCC CCGTTCCGGA GCTGCGTCCG TTCACGCTCA TCGCCGTGGA CG - #GTGAGGAC    1620    - CACCGCCGTC TGCGCCGCAT CCACGCACCG GCGTTCAACC CGCGCCGGCT GG - #CCGAGCGG    1680    - ACGGATCGCA TCGCCGCCAT CGCCGACCGG CTGCTCACCG AACTCGCCGA CT - #CCTCCGAC    1740    - CGGTCGGGCG AACCGGCCGA GCTGATCGGC GGCTTCGCGT ACCACTTCCC GC - #TGTTGGTC    1800    - ATCTGCGAAC TGCTCGGCGT GCCGGTCACC GATCCGGCAA TGGCCCGCGA GG - #CCGTCGGC    1860    - GTGCTCAAGG CACTCGGCCT CGGCGGCCCG CAGAGCGCCG GCGGTGACGG CA - #CGGACCCT    1920    - GCCGGGGACG TGCCGGACAC GTCGGCGCTG GAGAGCCTTC TCCTCGAAGC CG - #TGCACGCG    1980    - GCCCGGCGGA AAGACACCCG GACCATGACC CGCGTGCTCT ATGAACGCGC AC - #AGGCAGAG    2040    - TTCGGCTCGG TCTCCGACGA CCAGCTCGTC TACATGATCA CCGGACTCAT CT - #TCGCCGGC    2100    - CACGACACCA CCGGCTCGTT CCTGGGCTTC CTGCTTGCGG AGGTCCTGGC GG - #GCCGTCTC    2160    - GCGGCGGACG CCGACGGGGA CGCCATCTCC CGGTTCGTGG AGGAGGCGCT GC - #GCCACCAC    2220    - CCGCCGGTGC CCTACACGTT GTGGAGGTTC GCTGCCACGG AGGTGGTCAT CC - #GCGGTGTC    2280    - CGGCTGCCCC GCGGAGCGCC GGTACTGGTG GACATCGAGG GCACCAACAC CG - #ACGGCCGC    2340    - CATCACGACG CCCCGCACGC TTTCCACCCG GACCGCCCTT CGAGGCGGCG GC - #TCACCTTC    2400    - GGCGACGGGC CGCACTACTG CATCGGGGAG CAGCTCGCCC AGCTGGAATC GC - #GCACGATG    2460    - ATCGGCGTAC TGCGCAGCAG GTTCCCCCAA GCCCGACTGG CCGTGCCGTA CG - #AGGAGTTG    2520    - CGGTGGTGCA GGAAGGGGGC CCAGACAGCG CGGCTCACTG ACCTGCCCGT CT - #GGCTGCGT    2580    - TGATGGGCCG ACCGCGACCC GGCACGGGAC CGCCCACCGC CCATCGCGCG GT - #GGGCGGTC    2640    - CCGTGCCGGT CGCCCGGTGC GGTCCTCTCC CGACGCTCGC TCCCCCTGTG AC - #TTTCTCAC    2700    - ATCGAGACGT GACGAAATAA TCCCAGCAAG TGCCATGCAC ACTTTCATGG CG - #GACATTCA    2760    - CTTGCGAGGA TGGAGTGAGC ACACGGGGCC GCCCGAGACA CCCTACGGCC GC - #CGGAAGTA    2820    - TGCCACCTGT TGACGCGAAT GGAACGCCAC AGAGGGAGCA CCGCCAATGC AG - #ATCAATAT    2880    - GTTGGGCCCG CTCGTTGCAC ATCACAATGG CACGTCGGTG ACCCCGATAG CC - #AGAAAACC    2940    - CCGGCAGGTA TTCTCACTGC TCGCTCTTCA GGCAGGAACC GTCGTTCCGG TC - #CCCGCGCT    3000    #    3013    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2081 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 227..1649    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - TGTTGACAAT TAATCATCCG GCTCGTATAA TGTGTGGAAT TGTGAGCGGA TA - #ACAATTTC      60    - ACACAGGAAA CAGCGCCGCT GAGAAAAAGC GAAGCGGCAC TGCTCTTTAA CA - #ATTTATCA     120    - GACAATCTGT GTGGGCACTC GACCGGAATT GGGCATCGAT TAACTTTATT AT - #TAAAAATT     180    #GGG GGT       235AATGT ATCGATTAAA TAAGGAGGAA TAAACC ATG    #Gly            Met Gly    #                 1    - TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AG - #C ATG ACT GGT GGA CAG     283    Ser His His His His His His Gly Met Ala Se - #r Met Thr Gly Gly Gln    #      15    - CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GA - #T AAG GAT CGA TGG ATC     331    Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp As - #p Lys Asp Arg Trp Ile    # 35    - CGA CCT CGA GAT CTG CAG ATG GTA CCA TAT GG - #G AAT TCG GAG GGG TGC     379    Arg Pro Arg Asp Leu Gln Met Val Pro Tyr Gl - #y Asn Ser Glu Gly Cys    #                 50    - CTC ATG AGC GGC GAG GCG CCG CGG GTG GCC GT - #C GAC CCG TTC TCG TGT     427    Leu Met Ser Gly Glu Ala Pro Arg Val Ala Va - #l Asp Pro Phe Ser Cys    #             65    - CCC ATG ATG ACC ATG CAG CGC AAA CCC GAG GT - #G CAC GAC GCA TTC CGA     475    Pro Met Met Thr Met Gln Arg Lys Pro Glu Va - #l His Asp Ala Phe Arg    #         80    - GAG GCG GGC CCC GTC GTC GAG GTG AAC GCC CC - #C GCG GGC GGA CCC GCC     523    Glu Ala Gly Pro Val Val Glu Val Asn Ala Pr - #o Ala Gly Gly Pro Ala    #     95    - TGG GTC ATC ACC GAT GAC GCC CTC GCC CGC GA - #G GTG CTG GCC GAT CCC     571    Trp Val Ile Thr Asp Asp Ala Leu Ala Arg Gl - #u Val Leu Ala Asp Pro    100                 1 - #05                 1 - #10                 1 -    #15    - CGG TTC GTG AAG GGA CCC GAT CTC GCG CCC AC - #C GCC TGG CGG GGG GTG     619    Arg Phe Val Lys Gly Pro Asp Leu Ala Pro Th - #r Ala Trp Arg Gly Val    #               130    - GAC GAC GGT CTC GAC ATC CCC GTT CCG GAG CT - #G CGT CCG TTC ACG CTC     667    Asp Asp Gly Leu Asp Ile Pro Val Pro Glu Le - #u Arg Pro Phe Thr Leu    #           145    - ATC GCC GTG GAC GGT GAG GAC CAC CGG CGT CT - #G CGC CGC ATC CAC GCA     715    Ile Ala Val Asp Gly Glu Asp His Arg Arg Le - #u Arg Arg Ile His Ala    #       160    - CCG GCG TTC AAC CCG CGC CGG CTG GCC GAG CG - #G ACG GAT CGC ATC GCC     763    Pro Ala Phe Asn Pro Arg Arg Leu Ala Glu Ar - #g Thr Asp Arg Ile Ala    #   175    - GCC ATC GCC GAC CGG CTG CTC ACC GAA CTC GC - #C GAC TCC TCC GAC CGG     811    Ala Ile Ala Asp Arg Leu Leu Thr Glu Leu Al - #a Asp Ser Ser Asp Arg    180                 1 - #85                 1 - #90                 1 -    #95    - TCG GGC GAA CCG GCC GAG CTG ATC GGC GGC TT - #C GCG TAC CAC TTC CCG     859    Ser Gly Glu Pro Ala Glu Leu Ile Gly Gly Ph - #e Ala Tyr His Phe Pro    #               210    - CTG TTG GTC ATC TGC GAA CTG CTC GGC GTG CC - #G GTC ACC GAT CCG GCA     907    Leu Leu Val Ile Cys Glu Leu Leu Gly Val Pr - #o Val Thr Asp Pro Ala    #           225    - ATG GCC CGC GAG GCC GTC GGC GTG CTC AAG GC - #A CTC GGC CTC GGC GGC     955    Met Ala Arg Glu Ala Val Gly Val Leu Lys Al - #a Leu Gly Leu Gly Gly    #       240    - CCG CAG AGC GCC GGC GGT GAC GGC ACG GAC CC - #T GCC GGG GAC GTG CCG    1003    Pro Gln Ser Ala Gly Gly Asp Gly Thr Asp Pr - #o Ala Gly Asp Val Pro    #   255    - GAC ACG TCG GCG CTG GAG AGC CTT CTC CTC GA - #A GCC GTG CAC GCG GCC    1051    Asp Thr Ser Ala Leu Glu Ser Leu Leu Leu Gl - #u Ala Val His Ala Ala    260                 2 - #65                 2 - #70                 2 -    #75    - CGG CGG AAA GAC ACC CGG ACC ATG ACC CGC GT - #G CTC TAT GAA CGC GCA    1099    Arg Arg Lys Asp Thr Arg Thr Met Thr Arg Va - #l Leu Tyr Glu Arg Ala    #               290    - CAG GCA GAG TTC GGC TCG GTC TCC GAC GAC CA - #G CTC GTC TAC ATG ATC    1147    Gln Ala Glu Phe Gly Ser Val Ser Asp Asp Gl - #n Leu Val Tyr Met Ile    #           305    - ACC GGA CTC ATC TTC GCC GGC CAC GAC ACC AC - #C GGC TCG TTC CTG GGC    1195    Thr Gly Leu Ile Phe Ala Gly His Asp Thr Th - #r Gly Ser Phe Leu Gly    #       320    - TTC CTG CTT GCG GAG GTC CTG GCG GGC CGT CT - #C GCG GCG GAC GCC GAC    1243    Phe Leu Leu Ala Glu Val Leu Ala Gly Arg Le - #u Ala Ala Asp Ala Asp    #   335    - GGG GAC GCC ATC TCC CGG TTC GTG GAG GAG GC - #G CTG CGC CAC CAC CCG    1291    Gly Asp Ala Ile Ser Arg Phe Val Glu Glu Al - #a Leu Arg His His Pro    340                 3 - #45                 3 - #50                 3 -    #55    - CCG GTG CCC TAC TCG TTG TGG AGG TTC GCT GC - #C ACG GAG GTG GTC ATC    1339    Pro Val Pro Tyr Ser Leu Trp Arg Phe Ala Al - #a Thr Glu Val Val Ile    #               370    - CGC GGT GTC CGG CTG CCC CGC GGA GCG CCG GT - #A CTG GTG GAC ATC GAG    1387    Arg Gly Val Arg Leu Pro Arg Gly Ala Pro Va - #l Leu Val Asp Ile Glu    #           385    - GGC ACC AAC ACC GAC GGC CGC CAT CAC GAC GC - #C CCG CAC GCT TTC CAC    1435    Gly Thr Asn Thr Asp Gly Arg His His Asp Al - #a Pro His Ala Phe His    #       400    - CCG GAC CGC CCT TCG AGG CGG CGG CTC ACC TT - #C GGC GAC GGG CCG CAC    1483    Pro Asp Arg Pro Ser Arg Arg Arg Leu Thr Ph - #e Gly Asp Gly Pro His    #   415    - TAC TGC ATC GGG GAG CAG CTC GCC CAG CTG GA - #A TCG CGC ACG ATG ATC    1531    Tyr Cys Ile Gly Glu Gln Leu Ala Gln Leu Gl - #u Ser Arg Thr Met Ile    420                 4 - #25                 4 - #30                 4 -    #35    - GGC GTA CTG CGC AGC AGG TTC CCC CAA GCC CG - #A CTG GCC GTG CCG TAC    1579    Gly Val Leu Arg Ser Arg Phe Pro Gln Ala Ar - #g Leu Ala Val Pro Tyr    #               450    - GAG GAG TTG CGG TGG TGC AGG AAG GGG GCC CA - #G ACA GCG CGG CTC ACT    1627    Glu Glu Leu Arg Trp Cys Arg Lys Gly Ala Gl - #n Thr Ala Arg Leu Thr    #           465    - GAC CTG CCC GTC TGG CTG CGT T GATGGGCCGA CCGC - #GACCCG GCACGGGACC    1679    Asp Leu Pro Val Trp Leu Arg            470    - GCCCACCGCC CATCGCGCGG TGGGCGGTCC CGTGCCGGTC GCCCGGTGCG GT - #CCTCTCCC    1739    - GACGCTCGCT CCCCCTGTGA CTTTCTCACA TCGAGACGTG ACGAAATAAT CC - #CAGCAAGT    1799    - GCCATGCACA CTTTCATGGC GGACATTCAC TTGCGAGGAT GGAGTGAGCA CA - #CGGGGCCG    1859    - CCCGAGACAC CCTACGGCCG CCGGAAGTAT GCCACCTGTT GACGCGAATG GA - #ACGCCACA    1919    - GAGGGAGCAC CGGCAATGCA GATCAATATG TTGGGCCCGC TCGTTGCACA TC - #ACAATGGC    1979    - ACGTCGGTGA CCCCGATAGC CAGAAAACCC CGGCAGGTAT TCTCACTGCT CG - #CTCTTCAG    2039    #2081              CGGT CCCCGCGCTG ATGGAGGAGC TC    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 474 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Met Gly Gly Ser His His His His His His Gl - #y Met Ala Ser Met Thr    #                 15    - Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr As - #p Asp Asp Asp Lys Asp    #             30    - Arg Trp Ile Arg Pro Arg Asp Leu Gln Met Va - #l Pro Tyr Gly Asn Ser    #         45    - Glu Gly Cys Leu Met Ser Gly Glu Ala Pro Ar - #g Val Ala Val Asp Pro    #     60    - Phe Ser Cys Pro Met Met Thr Met Gln Arg Ly - #s Pro Glu Val His Asp    # 80    - Ala Phe Arg Glu Ala Gly Pro Val Val Glu Va - #l Asn Ala Pro Ala Gly    #                 95    - Gly Pro Ala Trp Val Ile Thr Asp Asp Ala Le - #u Ala Arg Glu Val Leu    #           110    - Ala Asp Pro Arg Phe Val Lys Gly Pro Asp Le - #u Ala Pro Thr Ala Trp    #       125    - Arg Gly Val Asp Asp Gly Leu Asp Ile Pro Va - #l Pro Glu Leu Arg Pro    #   140    - Phe Thr Leu Ile Ala Val Asp Gly Glu Asp Hi - #s Arg Arg Leu Arg Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Ile His Ala Pro Ala Phe Asn Pro Arg Arg Le - #u Ala Glu Arg Thr Asp    #               175    - Arg Ile Ala Ala Ile Ala Asp Arg Leu Leu Th - #r Glu Leu Ala Asp Ser    #           190    - Ser Asp Arg Ser Gly Glu Pro Ala Glu Leu Il - #e Gly Gly Phe Ala Tyr    #       205    - His Phe Pro Leu Leu Val Ile Cys Glu Leu Le - #u Gly Val Pro Val Thr    #   220    - Asp Pro Ala Met Ala Arg Glu Ala Val Gly Va - #l Leu Lys Ala Leu Gly    225                 2 - #30                 2 - #35                 2 -    #40    - Leu Gly Gly Pro Gln Ser Ala Gly Gly Asp Gl - #y Thr Asp Pro Ala Gly    #               255    - Asp Val Pro Asp Thr Ser Ala Leu Glu Ser Le - #u Leu Leu Glu Ala Val    #           270    - His Ala Ala Arg Arg Lys Asp Thr Arg Thr Me - #t Thr Arg Val Leu Tyr    #       285    - Glu Arg Ala Gln Ala Glu Phe Gly Ser Val Se - #r Asp Asp Gln Leu Val    #   300    - Tyr Met Ile Thr Gly Leu Ile Phe Ala Gly Hi - #s Asp Thr Thr Gly Ser    305                 3 - #10                 3 - #15                 3 -    #20    - Phe Leu Gly Phe Leu Leu Ala Glu Val Leu Al - #a Gly Arg Leu Ala Ala    #               335    - Asp Ala Asp Gly Asp Ala Ile Ser Arg Phe Va - #l Glu Glu Ala Leu Arg    #           350    - His His Pro Pro Val Pro Tyr Ser Leu Trp Ar - #g Phe Ala Ala Thr Glu    #       365    - Val Val Ile Arg Gly Val Arg Leu Pro Arg Gl - #y Ala Pro Val Leu Val    #   380    - Asp Ile Glu Gly Thr Asn Thr Asp Gly Arg Hi - #s His Asp Ala Pro His    385                 3 - #90                 3 - #95                 4 -    #00    - Ala Phe His Pro Asp Arg Pro Ser Arg Arg Ar - #g Leu Thr Phe Gly Asp    #               415    - Gly Pro His Tyr Cys Ile Gly Glu Gln Leu Al - #a Gln Leu Glu Ser Arg    #           430    - Thr Met Ile Gly Val Leu Arg Ser Arg Phe Pr - #o Gln Ala Arg Leu Ala    #       445    - Val Pro Tyr Glu Glu Leu Arg Trp Cys Arg Ly - #s Gly Ala Gln Thr Ala    #   460    - Arg Leu Thr Asp Leu Pro Val Trp Leu Arg    465                 4 - #70    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 443 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Asp Arg Trp Ile Arg Pro Arg Asp Leu Gln Me - #t Val Pro Tyr Gly Asn    #                15    - Ser Glu Gly Cys Leu Met Ser Gly Glu Ala Pr - #o Arg Val Ala Val Asp    #            30    - Pro Phe Ser Cys Pro Met Met Thr Met Gln Ar - #g Lys Pro Glu Val His    #        45    - Asp Ala Phe Arg Glu Ala Gly Pro Val Val Gl - #u Val Asn Ala Pro Ala    #    60    - Gly Gly Pro Ala Trp Val Ile Thr Asp Asp Al - #a Leu Ala Arg Glu Val    #80    - Leu Ala Asp Pro Arg Phe Val Lys Asp Pro As - #p Leu Ala Pro Thr Ala    #                95    - Trp Arg Gly Val Asp Asp Gly Leu Asp Ile Pr - #o Val Pro Glu Leu Arg    #           110    - Pro Phe Thr Leu Ile Ala Val Asp Gly Glu As - #p His Arg Arg Leu Arg    #       125    - Arg Ile His Ala Pro Ala Phe Asn Pro Arg Ar - #g Leu Ala Glu Arg Thr    #   140    - Asp Arg Ile Ala Ala Ile Ala Asp Arg Leu Le - #u Thr Glu Leu Ala Asp    145                 1 - #50                 1 - #55                 1 -    #60    - Ser Ser Asp Arg Ser Gly Glu Pro Ala Glu Le - #u Ile Gly Gly Phe Ala    #               175    - Tyr His Phe Pro Leu Leu Val Ile Cys Glu Le - #u Leu Gly Val Pro Val    #           190    - Thr Asp Pro Ala Met Ala Arg Glu Ala Val Gl - #y Val Leu Lys Ala Leu    #       205    - Gly Leu Gly Gly Pro Gln Ser Ala Gly Gly As - #p Gly Thr Asp Pro Ala    #   220    - Gly Asp Val Pro Asp Thr Ser Ala Leu Glu Se - #r Leu Leu Leu Glu Ala    225                 2 - #30                 2 - #35                 2 -    #40    - Val His Ala Ala Arg Arg Lys Asp Thr Arg Th - #r Met Thr Arg Val Leu    #               255    - Tyr Glu Arg Ala Gln Ala Glu Phe Gly Ser Va - #l Ser Asp Asp Gln Leu    #           270    - Val Tyr Met Ile Thr Gly Leu Ile Phe Ala Gl - #y His Asp Thr Thr Gly    #       285    - Ser Phe Leu Gly Phe Leu Leu Ala Glu Val Le - #u Ala Gly Arg Leu Ala    #   300    - Ala Asp Ala Asp Gly Asp Ala Ile Ser Arg Ph - #e Val Glu Glu Ala Leu    305                 3 - #10                 3 - #15                 3 -    #20    - Arg His His Pro Pro Val Pro Tyr Thr Leu Tr - #p Arg Phe Ala Ala Thr    #               335    - Glu Val Val Ile Arg Gly Val Arg Leu Pro Ar - #g Gly Ala Pro Val Leu    #           350    - Val Asp Ile Glu Gly Thr Asn Thr Asp Gly Ar - #g His His Asp Ala Pro    #       365    - His Ala Phe His Pro Asp Arg Pro Ser Arg Ar - #g Arg Leu Thr Phe Gly    #   380    - Asp Gly Pro His Tyr Cys Ile Gly Glu Gln Le - #u Ala Gln Leu Glu Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Arg Thr Met Ile Gly Val Leu Arg Ser Arg Ph - #e Pro Gln Ala Arg Leu    #               415    - Ala Val Pro Tyr Glu Glu Leu Arg Trp Cys Ar - #g Lys Gly Ala Gln Thr    #           430    - Ala Arg Leu Thr Asp Leu Pro Val Trp Leu Ar - #g    #       440    __________________________________________________________________________

What is claimed is:
 1. An isolated daunomycin C-14 hydroxylase whichconverts daunomycin to doxorubicin, wherein said daunomycin C-14hydroxylase is from Streptomyces sp. strain C5.
 2. The protein of claim1 further comprising a leader sequence encoding six histidine residues.3. The isolated daunomycin C-14 hydroxylase of claim 1, wherein saiddaunomycin C-hydroxylase has the amino acid sequence set forth in SEQ IDNO:5.
 4. The isolated daunomocyin C-14 hydroxylase of claim 3 furthercomprising a leader sequence encoding six histidine residues.